Induced stem cells (iSC) are
stem cells
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...
derived from
somatic
Somatic may refer to:
* Somatic (biology), referring to the cells of the body in contrast to the germ line cells
** Somatic cell, a non-gametic cell in a multicellular organism
* Somatic nervous system, the portion of the vertebrate nervous sys ...
,
reproductive
The reproductive system of an organism, also known as the genital system, is the biological system made up of all the anatomical organs involved in sexual reproduction. Many non-living substances such as fluids, hormones, and pheromones are als ...
,
pluripotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta.
According to Latin terms, Pluripotentia means the ability for many thin ...
or other cell types by deliberate
epigenetic
In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
reprogramming. They are classified as either
totipotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta.
According to Latin terms, Pluripotentia means the ability for many thin ...
(iTC),
pluripotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta.
According to Latin terms, Pluripotentia means the ability for many thin ...
(iPSC) or
progenitor
In genealogy, the progenitor (rarer: primogenitor; german: Stammvater or ''Ahnherr'') is the – sometimes legendary – founder of a family, line of descent, clan or tribe, noble house, or ethnic group..
Ebenda''Ahnherr:''"Stammvater eines G ...
(multipotent – iMSC, also called an induced multipotent progenitor cell – iMPC) or
unipotent
In mathematics, a unipotent element ''r'' of a ring ''R'' is one such that ''r'' − 1 is a nilpotent element; in other words, (''r'' − 1)''n'' is zero for some ''n''.
In particular, a square matrix ''M'' is a unipoten ...
– (iUSC) according to their
developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called
direct reprogramming or directed
differentiation and are also called induced
somatic stem cells.
Three techniques are widely recognized:
*
Transplantation of nuclei taken from somatic cells into an oocyte (egg cell) lacking its own nucleus (removed in lab)
*
Fusion
Fusion, or synthesis, is the process of combining two or more distinct entities into a new whole.
Fusion may also refer to:
Science and technology Physics
*Nuclear fusion, multiple atomic nuclei combining to form one or more different atomic nucl ...
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
A frog is any member of a diverse and largely Carnivore, carnivorous group of short-bodied, tailless amphibians composing the order (biology), order Anura (ανοὐρά, literally ''without tail'' in Ancient Greek). The oldest fossil "proto-f ...
's two
blastomere
In biology, a blastomere is a type of cell produced by cell division (cleavage) of the zygote after fertilization; blastomeres are an essential part of blastula formation, and blastocyst formation in mammals.
Human blastomere characteristics
In ...
s and found that
amphibians
Amphibians are four-limbed and ectothermic vertebrates of the class Amphibia. All living amphibians belong to the group Lissamphibia. They inhabit a wide variety of habitats, with most species living within terrestrial, fossorial, arbore ...
are able to form whole
embryo
An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...
s from the remaining part. This meant that the cells can change their differentiation pathway. In 1924 Spemann and Mangold demonstrated the key importance of cell–cell inductions during animal development. The reversible transformation of cells of one differentiated cell type to another is called
metaplasia
Metaplasia ( gr, "change in form") is the transformation of one differentiated cell type to another differentiated cell type. The change from one type of cell to another may be part of a normal maturation process, or caused by some sort of abno ...
. This transition can be a part of the normal maturation process, or caused by an inducement.
One example is the transformation of
iris
Iris most often refers to:
*Iris (anatomy), part of the eye
*Iris (mythology), a Greek goddess
* ''Iris'' (plant), a genus of flowering plants
* Iris (color), an ambiguous color term
Iris or IRIS may also refer to:
Arts and media
Fictional ent ...
cells to
lens
A lens is a transmissive optical device which focuses or disperses a light beam by means of refraction. A simple lens consists of a single piece of transparent material, while a compound lens consists of several simple lenses (''elements''), ...
cells in the process of maturation and transformation of
retinal pigment epithelium
The pigmented layer of retina or retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells, and is firmly attached to the underlying choroid and overlying retinal visual ce ...
cells into the neural retina during regeneration in adult
newt
A newt is a salamander in the subfamily Pleurodelinae. The terrestrial juvenile phase is called an eft. Unlike other members of the family Salamandridae, newts are semiaquatic, alternating between aquatic and terrestrial habitats. Not all aqua ...
eyes. This process allows the body to replace cells not suitable to new conditions with more suitable new cells. In
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
imaginal discs, cells have to choose from a limited number of standard discrete differentiation states. The fact that transdetermination (change of the path of differentiation) often occurs for a group of cells rather than single cells shows that it is induced rather than part of maturation.
The researchers were able to identify the minimal conditions and factors that would be sufficient for starting the cascade of molecular and cellular processes to instruct pluripotent cells to organize the
embryo
An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...
. They showed that
opposing gradients of
bone morphogenetic protein (BMP) and
Nodal
Nodal homolog is a secretory protein that in humans is encoded by the ''NODAL'' gene which is located on chromosome 10q22.1. It belongs to the transforming growth factor beta superfamily (TGF-β superfamily). Like many other members of this superf ...
, two
transforming growth factor Transforming growth factor (, or TGF) is used to describe two classes of polypeptide growth factors, TGFα and TGFβ.
The name "Transforming Growth Factor" is somewhat arbitrary, since the two classes of TGFs are not structurally or genetically ...
family members that act as
morphogen
A morphogen is a substance whose non-uniform distribution governs the pattern of tissue development in the process of morphogenesis or pattern formation, one of the core processes of developmental biology, establishing positions of the various ...
s, are sufficient to induce molecular and cellular mechanisms required to organize, ''
in vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
'' or ''
in vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
'',
uncommitted cells of the
zebrafish
The zebrafish (''Danio rerio'') is a freshwater fish belonging to the minnow family ( Cyprinidae) of the order Cypriniformes. Native to South Asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio (and thus often ...
blastula
Blastulation is the stage in early animal embryonic development that produces the blastula. In mammalian development the blastula develops into the blastocyst with a differentiated inner cell mass and an outer trophectoderm. The blastula (from ...
animal pole into a well-developed
embryo
An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...
.
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
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
.
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
endoderm
Endoderm is the innermost of the three primary germ layers in the very early embryo. The other two layers are the ectoderm (outside layer) and mesoderm (middle layer). Cells migrating inward along the archenteron form the inner layer of the gast ...
al,
ectoderm
The ectoderm is one of the three primary germ layers formed in early embryonic development. It is the outermost layer, and is superficial to the mesoderm (the middle layer) and endoderm (the innermost layer). It emerges and originates from t ...
al and mesodermal cells both in vitro and in vivo.
Other well-documented examples of
transdifferentiation
Transdifferentiation, also known as lineage reprogramming, is the process in which one mature somatic cell is transformed into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type. It is a type ...
and their significance in development and regeneration were described in detail.
Induced totipotent cells
SCNT-mediated
Induced totipotent cells can be obtained by reprogramming somatic cells with
somatic-cell nuclear transfer
In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory strategy for creating a viable embryo from a body cell and an egg cell. The technique consists of taking an enucleated oocyte (egg cell) and implantin ...
(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
Regenerative medicine deals with the "process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function". This field holds the promise of engineering damaged tissues and organs by st ...
. Unfortunately, the cells generated by this technology are potentially not completely protected from the
immune system
The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathogens, from viruses to parasitic worms, as well as cancer cells and objects such as wood splinte ...
of the patient (donor of nuclei), because they have the same
mitochondria
A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
l DNA, as a donor of oocytes, instead of the patients mitochondrial DNA. This reduces their value as a source for
autologous stem cell transplantation
Autologous stem-cell transplantation (also called autogenous, autogeneic, or autogenic stem-cell transplantation and abbreviated auto-SCT) is autologous transplantation of stem cells—that is, transplantation in which stem cells ( undifferentiat ...
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
Histone deacetylase inhibitors (HDAC inhibitors, HDACi, HDIs) are chemical compounds that inhibit histone deacetylases.
HDIs have a long history of use in psychiatry and neurology as mood stabilizers and anti-epileptics. More recently they are bei ...
, 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
Bioethics is both a field of study and professional practice, interested in ethical issues related to health (primarily focused on the human, but also increasingly includes animal ethics), including those emerging from advances in biology, med ...
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
Mitochondrial disease is a group of disorders caused by mitochondrial dysfunction. Mitochondria are the organelles that generate energy for the cell and are found in every cell of the human body except red blood cells. They convert the energy of ...
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
Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most euka ...
-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 (
trophectoderm
The trophoblast (from Greek : to feed; and : germinator) is the outer layer of cells of the blastocyst. Trophoblasts are present four days after fertilization in humans. They provide nutrients to the embryo and develop into a large part of the ...
al) 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
retrovirus
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...
es MuERV-L (MERVL).
Rejuvenation to iPSCs
![Healthy chimeric mouse from teratocarcinoma cells](https://upload.wikimedia.org/wikipedia/commons/e/e1/Healthy_chimeric_mouse_from_teratocarcinoma_cells.JPG)
iPSc were first obtained in the form of transplantable
teratocarcinoma
Germ cell tumor (GCT) is a neoplasm derived from germ cells. Germ-cell tumors can be cancerous or benign. Germ cells normally occur inside the gonads (ovary and testis). GCTs that originate outside the gonads may be birth defects resulting from er ...
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
Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre- implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consi ...
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
teratoma
A teratoma is a tumor made up of several different types of tissue, such as hair, muscle, teeth, or bone. Teratomata typically form in the ovary, testicle, or coccyx.
Symptoms
Symptoms may be minimal if the tumor is small. A testicular terato ...
s, 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
The inner cell mass (ICM) or embryoblast (known as the pluriblast in marsupials) is a structure in the early development of an embryo. It is the mass of cells inside the blastocyst that will eventually give rise to the definitive structures of ...
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
Niche may refer to:
Science
*Developmental niche, a concept for understanding the cultural context of child development
*Ecological niche, a term describing the relational position of an organism's species
*Niche differentiation, in ecology, the ...
").
In August 2006, Japanese researchers circumvented the need for an oocyte, as in SCNT. By reprograming mouse embryonic
fibroblast
A fibroblast is a type of cell (biology), biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework (Stroma (tissue), stroma) for animal Tissue (biology), tissues, and plays a critical role in wound ...
s into pluripotent stem cells via the ectopic expression of four
transcription factor
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 fu ...
s, namely
Oct4
Oct-4 (octamer-binding transcription factor 4), also known as POU5F1 (POU domain, class 5, transcription factor 1), is a protein that in humans is encoded by the ''POU5F1'' gene. Oct-4 is a homeodomain transcription factor of the POU family. I ...
,
Sox2,
Klf4 and
c-Myc
''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' (MYC), ''l-myc'' (MYCL), and ''n-myc'' (MYCN). ''c-myc'' (also sometimes referre ...
, 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.
![Overview of iPS cells](https://upload.wikimedia.org/wikipedia/commons/2/28/Overview_of_iPS_cells.png)
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
Polyploidy is a condition in which the cells of an organism have more than one pair of ( homologous) chromosomes. Most species whose cells have nuclei (eukaryotes) are diploid, meaning they have two sets of chromosomes, where each set contains ...
(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
Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre- implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consi ...
, 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
An immune response is a reaction which occurs within an organism for the purpose of defending against foreign invaders. These invaders include a wide variety of different microorganisms including viruses, bacteria, parasites, and fungi which could ...
, 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 isoform
A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isof ...
s. 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
Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
(via
cytochrome c
The cytochrome complex, or cyt ''c'', is a small hemeprotein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins and plays a major role in cell apoptosis. Cytochrome c is hig ...
release across the
mitochondria
A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
l 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
Within the fields of molecular biology and pharmacology, a small molecule or micromolecule is a low molecular weight (≤ 1000 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Many drugs ...
(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
Oleic acid is a fatty acid that occurs naturally in various animal and vegetable fats and oils. It is an odorless, colorless oil, although commercial samples may be yellowish. In chemical terms, oleic acid is classified as a monounsaturated omega ...
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
Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, is a protein that, in humans, is encoded by the ''BIRC5'' gene.
Survivin is a member of the inhibitor of apoptosis (IAP) family. The survivin protein functions ...
or Bcl10). A single treatment with chemical survivin inhibitors (e.g.,
quercetin
Quercetin is a plant flavonol from the flavonoid group of polyphenols. It is found in many fruits, vegetables, leaves, seeds, and grains; capers, red onions, and kale are common foods containing appreciable amounts of it. It has a bitter flavor a ...
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.
![Stem cell modulators](https://upload.wikimedia.org/wikipedia/commons/e/eb/Stem_cell_modulators.svg)
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
Polycomb-group proteins (PcG proteins) are a family of protein complexes first discovered in fruit flies that can remodel chromatin such that epigenetic silencing of genes takes place. Polycomb-group proteins are well known for silencing Hox genes ...
targets and altered
DNA methylation
DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts t ...
) 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".
Chemical inducement
By using solely
small molecules
Within the fields of molecular biology and pharmacology, a small molecule or micromolecule is a low molecular weight (≤ 1000 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Many drugs ...
, 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
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of Genetic engineering techniques, technologies used to change the gene ...
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
A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Monoclonal antibodies ca ...
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
Programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the ''CD274'' gene.
Programmed death-ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protei ...
, which activates T cell inhibitory pathway.
See also: .
Differentiated cell types
Retinal cells
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
Chronic obstructive pulmonary disease (COPD) is a type of progressive lung disease characterized by long-term respiratory symptoms and airflow limitation. The main symptoms include shortness of breath and a cough, which may or may not produce ...
and
asthma
Asthma is a long-term inflammatory disease of the airways of the lungs. It is characterized by variable and recurring symptoms, reversible airflow obstruction, and easily triggered bronchospasms. Symptoms include episodes of wheezing, cou ...
, 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
lung
The lungs are the primary organs of the respiratory system in humans and most other animals, including some snails and a small number of fish. In mammals and most other vertebrates, two lungs are located near the backbone on either side of t ...
tissue engineering
Tissue engineering is a biomedical engineering discipline that uses a combination of Cell (biology), cells, engineering, Materials science, materials methods, and suitable biochemistry, biochemical and physicochemical factors to restore, maintai ...
.
Several protocols have been developed for generation of most cell types of the
respiratory system
The respiratory system (also respiratory apparatus, ventilatory system) is a biological system consisting of specific organs and structures used for gas exchange in animals and plants. The anatomy and physiology that make this happen varies grea ...
, 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 The myogenic mechanism is how arteries and arterioles react to an increase or decrease of blood pressure to keep the blood flow constant within the blood vessel. Myogenic response refers to a contraction initiated by the myocyte itself instead of ...
, 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
Platelet-derived growth factor (PDGF) is one among numerous growth factors that regulate cell growth and division. In particular, PDGF plays a significant role in blood vessel formation, the growth of blood vessels from already-existing blood v ...
–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
The pharynx (plural: pharynges) is the part of the throat behind the mouth and nasal cavity, and above the oesophagus and trachea (the tubes going down to the stomach and the lungs). It is found in vertebrates and invertebrates, though its struc ...
into fully differentiated intestinal cells in intact
larvae
A larva (; plural larvae ) is a distinct juvenile form many animals undergo before metamorphosis into adults. Animals with indirect development such as insects, amphibians, or cnidarians typically have a larval phase of their life cycle.
The ...
and adult roundworm ''
Caenorhabditis elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (ro ...
'' with no requirement for a dedifferentiated intermediate.
Transdifferentiation with CRISPR-mediated activator
The cell fate can be effectively manipulated by
epigenome editing
Epigenome editing or Epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites (as opposed to whole-genome modifications). Whereas gene editing inv ...
, in particular via the direct activation of specific endogenous gene expression with
CRISPR
CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacte ...
-mediated activator. When
dCas9
CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs).
Like for CRISPR interference, the CR ...
(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
Transcription factor GATA-6, also known as GATA-binding factor 6 (GATA6), is protein that in humans is encoded by the ''GATA6'' gene. The gene product preferentially binds (A/T/C)GAT(A/T)(A) of the consensus binding sequence.
Clinical significa ...
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
Amphibians are tetrapod, four-limbed and ectothermic vertebrates of the Class (biology), class Amphibia. All living amphibians belong to the group Lissamphibia. They inhabit a wide variety of habitats, with most species living within terres ...
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
Reversine, or 2-(4-morpholinoanilino)-6-cyclohexylaminopurine, is a small molecule developed by the group of Peter G. Schultz, used for stem cell dedifferentiation.
It also has the potential to selectively induce cell death in cancer cells.
Re ...
(the
aurora B kinase
Aurora kinase B is a protein that functions in the attachment of the mitotic spindle to the centromere.
Function
Chromosomal segregation during mitosis as well as meiosis is regulated by kinases and phosphatases. The Aurora kinases associate wi ...
inhibitor), and some other chemicals (BIO (glycogen synthase-3 kinase inhibitor),
lysophosphatidic acid
Lysophosphatidic acid (LPA) is a phospholipid derivative that can act as a signaling molecule.
Function
LPA acts as a potent mitogen due to its activation of three high-affinity G-protein-coupled receptors called LPAR1, LPAR2, and LPAR3 (a ...
(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
Granulocyte colony-stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.
Functi ...
-mimicking
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
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
Schlegel
Schlegel is a German occupational surname. Notable people with the surname include:
* Anthony Schlegel (born 1981), former American football linebacker
* August Wilhelm Schlegel (1767–1845), German poet, older brother of Friedrich
* Brad Schlege ...
and Liu
demonstrated that the combination of feeder cells
and a
Rho kinase
Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC (PKA/ PKG/PKC) family of serine-threonine specific protein kinases. It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton.
ROCKs ( ...
inhibitor (Y-27632)
induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro. This process occurs without the need for transduction of exogenous viral or cellular genes. These cells have been termed "Conditionally Reprogrammed Cells (CRC)". The induction of CRCs is rapid and results from reprogramming of the entire cell population. CRCs do not express high levels of proteins characteristic of iPSCs or embryonic stem cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of CRCs is reversible and removal of Y-27632 and feeders allows the cells to differentiate normally.
CRC technology can generate 2 cells in 5 to 6 days from needle biopsies and can generate cultures from cryopreserved tissue and from fewer than four viable cells. CRCs retain a normal
karyotype
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors.
The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking.
Using CRC technology, researchers were able to identify an effective therapy for a patient with a rare type of lung tumor.
Engleman's group
describes a pharmacogenomic platform that facilitates rapid discovery of drug combinations 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
CD47 (Cluster of Differentiation 47) also known as integrin associated protein (IAP) is a transmembrane protein that in humans is encoded by the CD47 gene. CD47 belongs to the immunoglobulin superfamily and partners with membrane integrins and ...
– a
membrane protein
Membrane proteins are common proteins that are part of, or interact with, biological membranes. Membrane proteins fall into several broad categories depending on their location. Integral membrane proteins are a permanent part of a cell membrane ...
that is the
thrombospondin-1
Thrombospondin 1, abbreviated as THBS1, is a protein that in humans is encoded by the ''THBS1'' gene.
Thrombospondin 1 is a subunit of a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein that mediates cell-to-cell an ...
receptor. Loss of CD47 permits sustained proliferation of primary
murine
The Old World rats and mice, part of the subfamily Murinae in the family Muridae, comprise at least 519 species. Members of this subfamily are called murines. In terms of species richness, this subfamily is larger than all mammal families excep ...
endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent
embryoid body
Embryoid bodies (EBs) are three-dimensional aggregates of pluripotent stem cells.
EBs are differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers.
Background
The pluripotent cell type ...
-like clusters. CD47 knockdown acutely increases
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
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
A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
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
Macrophages (abbreviated as M φ, MΦ or MP) ( el, large eaters, from Greek ''μακρός'' (') = large, ''φαγεῖν'' (') = to eat) are a type of white blood cell of the immune system that engulfs and digests pathogens, such as cancer ce ...
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
White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...
– which is usually not possible with
differentiated cells. The macrophages achieve this by activating a
gene network
A gene (or genetic) regulatory network (GRN) is a collection of molecular regulators that interact with each other and with other substances in the cell to govern the gene expression levels of mRNA and proteins which, in turn, determine the fu ...
similar to one found in embryonic stem cells.
Single-cell analysis
In the field of cellular biology, single-cell analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in th ...
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
Transcription factor MafB also known as V-maf musculoaponeurotic fibrosarcoma oncogene homolog B is a protein that in humans is encoded by the ''MAFB'' gene. This gene maps to chromosome 20q11.2-q13.1, consists of a single exon and spans around 3 ...
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
''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' (MYC), ''l-myc'' (MYCL), and ''n-myc'' (MYCN). ''c-myc'' (also sometimes referre ...
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
Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not al ...
of their outer membrane. That distinguishes them from most nonpluripotent cells, although not from
white blood cells
White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...
. The
glycan
The terms glycans and polysaccharides are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate p ...
s 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 marker Stem cell markers are genes and their protein products used by scientists to isolate and identify stem cells. Stem cells can also be identified by functional assays. Below is a list of genes/protein products that can be used to identify various typ ...
s are based on cell surface glycan epitopes, including the widely used markers
SSEA-3 Stage-specific embryonic antigen 3 (SSEA-3) is a glycosphingolipid, specifically, an oligosaccharide composed of five carbohydrate units connected to a sphingolipid. Sphingolipids were originally discovered in 1884 by Johann Ludwig Wilhelm Thudichum ...
, 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
The Notch signaling pathway is a highly Conserved sequence, conserved cell signaling system present in most animals. Mammals possess four different Notch proteins, notch receptors, referred to as NOTCH1, NOTCH2, Notch 3, NOTCH3, and NOTCH4. The ...
- 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
Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycos ...
ACA, linking glycosylphosphatidylinositol on the surface of the progenitor cells in human peripheral blood induces increased expression of genes
Wnt,
Notch-1
Neurogenic locus notch homolog protein 1 (Notch 1) is a protein encoded in humans by the ''NOTCH1'' gene. Notch 1 is a single-pass transmembrane receptor.
Function
This gene encodes a member of the Notch family. Members of this Type 1 transme ...
,
BMI1
Polycomb complex protein BMI-1 also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51) is a protein that in humans is encoded by the ''BMI1'' gene (B cell-specific Moloney murine leukemia virus integration ...
and
HOXB4
Homeobox protein Hox-B4 is a protein that in humans is encoded by the ''HOXB4'' gene.
Function
This gene is a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of h ...
through a signaling cascade
PI3K
Phosphoinositide 3-kinases (PI3Ks), also called phosphatidylinositol 3-kinases, are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which i ...
/
Akt
Protein kinase B (PKB), also known as Akt, is the collective name of a set of three serine/threonine-specific protein kinases that play key roles in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, tran ...
/
mTor
The mammalian target of sirolimus, rapamycin (mTOR), also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), is a kinase that in humans is encoded by the ''MT ...
/
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
A germ layer is a primary layer of cells that forms during embryonic development. The three germ layers in vertebrates are particularly pronounced; however, all eumetazoans (animals that are sister taxa to the sponges) produce two or three pri ...
.
The study of
lectin
Lectins are carbohydrate-binding proteins that are highly specific for sugar groups that are part of other molecules, so cause agglutination of particular cells or precipitation of glycoconjugates and polysaccharides. Lectins have a role in rec ...
s' ability to maintain a culture of pluripotent human stem cells has led to the discovery of lectin
Erythrina crista-galli
''Erythrina crista-galli'', often known as the cockspur coral tree, is a flowering tree in the family Fabaceae, native to Argentina, Uruguay, southern Brazil and Paraguay. It is widely planted as a street or garden tree in other countries, not ...
(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
ECM proteoglycan
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan (GAG) chain(s). The point of attachment is a serine (Ser) residue to whic ...
,
fibromodulin
Fibromodulin is a protein that in humans is encoded by the ''FMOD'' gene.
Fibromodulin is a 42kDa protein of a family of small interstitial leucine-rich repeat proteoglycans (SLRPs). It can have up to four N-linked keratan sulfate chains attach ...
. 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 protein E-cadherin
Cadherin-1 or Epithelial cadherin (E-cadherin), (not to be confused with the APC/C activator protein CDH1) is a protein that in humans is encoded by the ''CDH1'' gene. Mutations are correlated with gastric, breast, colorectal, thyroid, and ovarian ...
is indispensable for a robust pluripotent
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
. During reprogramming for iPS cell generation,
N-cadherin
Cadherin-2 also known as Neural cadherin (N-cadherin), is a protein that in humans is encoded by the ''CDH2'' gene. CDH2 has also been designated as CD325 (cluster of differentiation 325).
Cadherin-2 is a transmembrane protein expressed in multipl ...
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.
![Adhesion diagram](https://upload.wikimedia.org/wikipedia/commons/5/5d/Adhesion_diagram.jpg)
Substrate rigidity is an important biophysical cue influencing neural induction and subtype specification. For example, soft substrates promote neuroepithelial conversion while inhibiting
neural crest
Neural crest cells are a temporary group of cells unique to vertebrates that arise from the embryonic ectoderm germ layer, and in turn give rise to a diverse cell lineage—including melanocytes, craniofacial cartilage and bone, smooth muscle, per ...
differentiation of hESCs in a
BMP4
Bone morphogenetic protein 4 is a protein that in humans is encoded by ''BMP4'' gene. BMP4 is found on chromosome 14q22-q23.
BMP4 is a member of the bone morphogenetic protein family which is part of the transforming growth factor-beta superfamil ...
-dependent manner. Mechanistic studies revealed a multi-targeted mechanotransductive process involving mechanosensitive
Smad phosphorylation
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
and nucleocytoplasmic shuttling, regulated by rigidity-dependent
Hippo
The hippopotamus ( ; : hippopotamuses or hippopotami; ''Hippopotamus amphibius''), also called the hippo, common hippopotamus, or river hippopotamus, is a large semiaquatic mammal native to sub-Saharan Africa. It is one of only two extant ...
/
YAP
Yap ( yap, Waqaab) traditionally refers to an island group located in the Caroline Islands of the western Pacific Ocean, a part of Yap State. The name "Yap" in recent years has come to also refer to the state within the Federated States of Micr ...
activities and
actomyosin
Myofilaments are the three protein filaments of myofibrils in muscle cells. The main proteins involved are myosin, actin, and titin. Myosin and actin are the ''contractile proteins'' and titin is an elastic protein. The myofilaments act toget ...
cytoskeleton
The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is compos ...
integrity and
contractility
Contractility refers to the ability for self-contraction, especially of the muscles or similar active biological tissue
*Contractile ring in cytokinesis
*Contractile vacuole
*Muscle contraction
**Myocardial contractility
*See contractile cell for ...
.
Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the
cytokine
Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in autocrin ...
leukemia inhibitory factor
Leukemia inhibitory factor, or LIF, is an interleukin 6 class cytokine that affects cell growth by inhibiting differentiation. When LIF levels drop, the cells differentiate.
Function
LIF derives its name from its ability to induce the terminal ...
(LIF). Following LIF withdrawal, mESCs differentiate, accompanied by an increase in cell–substratum
adhesion
Adhesion is the tendency of dissimilar particles or surfaces to cling to one another ( cohesion refers to the tendency of similar or identical particles/surfaces to cling to one another).
The forces that cause adhesion and cohesion can be ...
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
The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is compos ...
, 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
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating wi ...
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 device
Microfluidics refers to the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small scale (typically sub-millimeter) at which surface forces dominate volumetric forces. It is a multidisciplinary field tha ...
s, 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
The Hippo signaling pathway, also known as the Salvador-Warts-Hippo (SWH) pathway, is a signaling pathway that controls organ size in animals through the regulation of cell proliferation and apoptosis. The pathway takes its name from one of its k ...
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 pathway
The transforming growth factor beta (TGFB) signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, cell migration, apoptosis, cellular homeostasis an ...
s), under a physiological
hypoxic condition.
Alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase and TGF-β pathways (where:
sodium butyrate
Sodium butyrate is a compound with formula Na(C3H7COO). It is the sodium salt of butyric acid. It has various effects on cultured mammalian cells including inhibition of proliferation, induction of differentiation and induction or repression ...
(NaB) or
Trichostatin A
Trichostatin A (TSA) is an organic compound that serves as an antifungal antibiotic and selectively inhibits the class I and II mammalian histone deacetylase (HDAC) families of enzymes, but not class III HDACs (i.e., sirtuins). However, there are ...
(TSA) could replace VPA,
Lithium chloride
Lithium chloride is a chemical compound with the formula Li Cl. The salt is a typical ionic compound (with certain covalent characteristics), although the small size of the Li+ ion gives rise to properties not seen for other alkali metal chlor ...
(LiCl) or lithium carbonate (Li2CO3) could substitute CHIR99021, or Repsox may be replaced with
SB-431542
SB-431542 is a drug candidate developed by GlaxoSmithKline (GSK) as an inhibitor of the activin receptor-like kinase (ALK) receptors, ALK5, ALK4 and ALK7. However, it is ''not'' an inhibitor of anaplastic lymphoma kinase (which are commonly kno ...
or
Tranilast
Tranilast (INN, brand name Rizaben) is an antiallergic drug. It was developed by Kissei Pharmaceuticals and was approved in 1982 for use in Japan and South Korea for bronchial asthma. Indications for keloid and hypertrophic scar were added in ...
) 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
astrocyte
Astrocytes (from Ancient Greek , , "star" + , , "cavity", "cell"), also known collectively as astroglia, are characteristic star-shaped glial cells in the brain and spinal cord. They perform many functions, including biochemical control of e ...
s directly in the brain that are engineered to express inducible forms of neural reprogramming genes, into neurons, when reprogramming genes (
Ascl1
Achaete-scute homolog 1 is a protein that in humans is encoded by the ''ASCL1'' gene. Because it was discovered subsequent to studies on its homolog in Drosophila, the Achaete-scute complex, it was originally named MASH-1 for mammalian achaete scu ...
,
Brn2a and
Myt1l) are activated after transplantation using a drug.
Astrocytes
Astrocytes (from Ancient Greek , , "star" + , , "cavity", "cell"), also known collectively as astroglia, are characteristic star-shaped glial cells in the brain and spinal cord. They perform many functions, including biochemical control of endo ...
– the most common
neuroglia
Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form mye ...
l brain cells, which contribute to
scar
A scar (or scar tissue) is an area of fibrous tissue that replaces normal skin after an injury. Scars result from the biological process of wound repair in the skin, as well as in other organs, and tissues of the body. Thus, scarring is a na ...
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
Myelin is a lipid-rich material that surrounds nerve cell axons (the nervous system's "wires") to insulate them and increase the rate at which electrical impulses (called action potentials) are passed along the axon. The myelinated axon can be ...
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
Oligodendrocytes (), or oligodendroglia, are a type of neuroglia whose main functions are to provide support and insulation to axons in the central nervous system of jawed vertebrates, equivalent to the function performed by Schwann cells in the ...
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
Cardiac muscle (also called heart muscle, myocardium, cardiomyocytes and cardiac myocytes) is one of three types of vertebrate muscle tissues, with the other two being skeletal muscle and smooth muscle. It is an involuntary, striated muscle th ...
-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
Ligation may refer to:
* Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules
* In medicine, the making of a ligature (tie)
* Chemical ligation, the production of peptides from amino acids
* Tubal ligation, a meth ...
.
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
Ischemic cardiomyopathy is a type of cardiomyopathy caused by a narrowing of the coronary arteries which supply blood to the heart. Typically, patients with ischemic cardiomyopathy have a history of acute myocardial infarction, however, it may occ ...
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
In medicine, decompensation is the functional deterioration of a structure or system that had been previously working with the help of compensation. Decompensation may occur due to fatigue, stress, illness, or old age. When a system is "compensate ...
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
The Wnt signaling pathways are a group of signal transduction pathways which begin with proteins that pass signals into a cell through cell surface receptors. The name Wnt is a portmanteau created from the names Wingless and Int-1. Wnt signaling p ...
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
Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-β 1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other sign ...
receptor type II and selectively inhibits intracellular TGF-β signaling. It thus selectively enhances the differentiation of uncommitted
mesoderm
The mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm.Langman's Medical E ...
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
SB-431542 is a drug candidate developed by GlaxoSmithKline (GSK) as an inhibitor of the activin receptor-like kinase (ALK) receptors, ALK5, ALK4 and ALK7. However, it is ''not'' an inhibitor of anaplastic lymphoma kinase (which are commonly kno ...
(the transforming growth factor (TGF)-β inhibitor), and XAV939 (the WNT inhibitor) for 2 weeks after myocardial infarction showed significantly improved reprogramming (reprogramming efficiency increased eight-fold) and cardiac function compared to those exposed to only GMT.
See also: review
Rejuvenation of the muscle stem cell
The elderly often with progressive
muscle weakness
Muscle weakness is a lack of muscle strength. Its causes are many and can be divided into conditions that have either true or perceived muscle weakness. True muscle weakness is a primary symptom of a variety of skeletal muscle diseases, includi ...
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
A hydrogel is a crosslinked hydrophilic polymer that does not dissolve in water. They are highly absorbent yet maintain well defined structures. These properties underpin several applications, especially in the biomedical area. Many hydrogels ar ...
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
p16
p16 (also known as p16INK4a, cyclin-dependent kinase inhibitor 2A, CDKN2A, multiple tumor suppressor 1 and numerous other synonyms), is a protein that slows cell division by slowing the progression of the cell cycle from the G1 phase to the S p ...
INK4a (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
Fibroblast growth factor 2, also known as basic fibroblast growth factor (bFGF) and FGF-β, is a growth factor and signaling protein encoded by the ''FGF2'' gene. It binds to and exerts effects via specific fibroblast growth factor receptor ''(FG ...
) and
epidermal growth factor
Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. Human EGF is 6-k Da and has 53 amino acid residues and three intramolecular disulfide bonds.
EGF was originally descr ...
.
Hepatocytes
Unlike current protocols for deriving
hepatocyte
A hepatocyte is a cell of the main parenchymal tissue of the liver. Hepatocytes make up 80% of the liver's mass.
These cells are involved in:
* Protein synthesis
* Protein storage
* Transformation of carbohydrates
* Synthesis of cholesterol, ...
s 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.
![Gray1062](https://upload.wikimedia.org/wikipedia/commons/e/e5/Gray1062.png)
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
Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, glycogen ...
), 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
Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ap ...
such as
cardiovascular diseases
Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels. CVD includes coronary artery diseases (CAD) such as angina and myocardial infarction (commonly known as a heart attack). Other CVDs include stroke, ...
,
retinopathy
Retinopathy is any damage to the retina of the eyes, which may cause vision impairment. Retinopathy often refers to retinal vascular disease, or damage to the retina caused by abnormal blood flow. Age-related macular degeneration is technically inc ...
,
neuropathy
Peripheral neuropathy, often shortened to neuropathy, is a general term describing disease affecting the peripheral nerves, meaning nerves beyond the brain and spinal cord. Damage to peripheral nerves may impair sensation, movement, gland, or o ...
,
nephropathy
Kidney disease, or renal disease, technically referred to as nephropathy, is damage to or disease of a kidney. Nephritis is an inflammatory kidney disease and has several types according to the location of the inflammation. Inflammation can ...
, and peripheral circulatory diseases depend on
sugar dysregulation due to lack of
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
from pancreatic
beta cells
Beta cells (β-cells) are a type of cell found in pancreatic islets that synthesize and secrete insulin and amylin. Beta cells make up 50–70% of the cells in human islets. In patients with Type 1 diabetes, beta-cell mass and function are dimini ...
and can be lethal if they are not treated. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs).
Unfortunately, human PSC-derived insulin-expressing cells resemble human fetal β cells rather than adult β cells. In contrast to adult β cells, fetal β cells seem functionally immature, as indicated by increased
basal glucose
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using ...
secretion and lack of glucose stimulation and confirmed by
RNA-seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
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 factor
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 fu ...
s,
PDX1
PDX1 (pancreatic and duodenal homeobox 1), also known as insulin promoter factor 1, is a transcription factor in the ParaHox gene cluster.Brooke, N. M., Garcia-Fernàndez, J., & Holland, P. W. (1998). The ParaHox gene cluster is an evolutionary si ...
(required for
pancreatic bud outgrowth and beta-cell maturation),
NGN3
Neurogenins are a family of bHLH transcription factors involved in specifying neuronal differentiation. It is one of many gene families related to the ''atonal'' gene in Drosophila. Other positive regulators of neuronal differentiation also ex ...
(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
The gastrointestinal tract (GI tract, digestive tract, alimentary canal) is the tract or passageway of the digestive system that leads from the mouth to the anus. The GI tract contains all the major organs of the digestive system, in humans ...
. PMN expression in human intestinal "
organoid
An organoid is a miniaturized and simplified version of an Organ (anatomy), organ produced in vitro in three dimensions that shows realistic micro-anatomy. They are derived from one or a few Cell (biology), cells from a Tissue (biology), tissue, ...
s" 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
The nephron is the minute or microscopic structural and functional unit of the kidney. It is composed of a renal corpuscle and a renal tubule. The renal corpuscle consists of a tuft of capillaries called a glomerulus and a cup-shaped structure ca ...
progenitors of the embryonic
kidney
The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blood ...
, 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
Mesenchyme () is a type of loosely organized animal embryonic connective tissue of undifferentiated cells that give rise to most tissues, such as skin, blood or bone. The interactions between mesenchyme and epithelium help to form nearly every o ...
/nephron progenitor phenotype in the adult proximal tubule cell line.
The generation of such cells may lead to cellular therapies for adult
renal disease
Kidney disease, or renal disease, technically referred to as nephropathy, is damage to or disease of a kidney. Nephritis is an inflammatory kidney disease and has several types according to the location of the inflammation. Inflammation can b ...
. 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
Collateral circulation is the alternate circulation around a blocked artery or vein via another path, such as nearby minor vessels. It may occur via preexisting vascular redundancy (analogous to engineered redundancy), as in the circle of Wil ...
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
Cardiac muscle (also called heart muscle, myocardium, cardiomyocytes and cardiac myocytes) is one of three types of vertebrate muscle tissues, with the other two being skeletal muscle and smooth muscle. It is an involuntary, striated muscle that ...
, 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
Protein kinase B (PKB), also known as Akt, is the collective name of a set of three serine/threonine-specific protein kinases that play key roles in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, tran ...
, which positively regulates neovasculogenesis) of
bone marrow
Bone marrow is a semi-solid tissue found within the spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It is composed of hematopoietic ce ...
–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
Liposuction, or simply lipo, is a type of fat-removal procedure used in plastic surgery. Evidence does not support an effect on weight beyond a couple of months and does not appear to affect obesity-related problems. In the United States, lipo ...
may be coaxed into becoming progenitor
smooth muscle
Smooth muscle is an involuntary non-striated muscle, so-called because it has no sarcomeres and therefore no striations (''bands'' or ''stripes''). It is divided into two subgroups, single-unit and multiunit smooth muscle. Within single-unit mus ...
cells (iPVSMCs) found in arteries and veins.
[
*]
The 2D culture system of human iPS cells
in conjunction with triple marker selection (
CD34
CD34 is a transmembrane phosphoglycoprotein protein encoded by the CD34 gene in humans, mice, rats and other species.
CD34 derives its name from the cluster of differentiation protocol that identifies cell surface antigens. CD34 was first descri ...
(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 Paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as paracrine factors diffuse ove ...
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
Vascular endothelial growth factor A (VEGF-A) is a protein that in humans is encoded by the ''VEGFA'' gene.
Function
This gene is a member of the platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) family and encod ...
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
CD34 is a transmembrane phosphoglycoprotein protein encoded by the CD34 gene in humans, mice, rats and other species.
CD34 derives its name from the cluster of differentiation protocol that identifies cell surface antigens. CD34 was first descri ...
positive cells have normal survival when transfused into an autologous recipient.
RBC produced in vitro contained exclusively
fetal hemoglobin
Fetal hemoglobin, or foetal haemoglobin (also hemoglobin F, HbF, or α2γ2) is the main oxygen carrier protein in the human fetus. Hemoglobin F is found in fetal red blood cells, and is involved in transporting oxygen from the mother's bloodstream ...
(HbF), which rescues the functionality of these RBCs. In vivo the switch of fetal to adult hemoglobin was observed after infusion of nucleated
erythroid
Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "hol ...
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
In organic chemistry, an aryl is any functional group or substituent derived from an aromatic ring, usually an aromatic hydrocarbon, such as phenyl and naphthyl. "Aryl" is used for the sake of abbreviation or generalization, and "Ar" is used as ...
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
SH2B adapter protein 3 (SH2B3), also known as lymphocyte adapter protein (LNK), is a protein that in humans is encoded by the ''SH2B3'' gene on chromosome 12.
SH2B adapter protein 3 is a protein that in humans is encoded by the SH2B3 gene on chrom ...
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
CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacte ...
/
Cas9
Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic e ...
genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation.
(See also overview.
)
Platelets
Platelets
Platelets, also called thrombocytes (from Greek θρόμβος, "clot" and κύτος, "cell"), are a component of blood whose function (along with the coagulation factors) is to react to bleeding from blood vessel injury by clumping, thereby ini ...
help prevent hemorrhage in
thrombocytopenic
Thrombocytopenia is a condition characterized by abnormally low levels of platelets, also known as thrombocytes, in the blood. It is the most common coagulation disorder among intensive care patients and is seen in a fifth of medical patients an ...
patients and patients with
thrombocythemia
Thrombocythemia is a condition of high platelet (thrombocyte) count in the blood. Normal count is in the range of 150x109 to 450x109 platelets per liter of blood, but investigation is typically only considered if the upper limit exceeds 750x109/L. ...
. 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
RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by o ...
-based mechanism used a
lentiviral vector
Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cell (biology), cells. This process can be performed inside a living organism (''in vivo'') or in cell culture (''in vitro''). Viruses have evolved spec ...
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
Doxycycline is a broad-spectrum tetracycline class antibiotic used in the treatment of infections caused by bacteria and certain parasites. It is used to treat bacterial pneumonia, acne, chlamydia infections, Lyme disease, cholera, typhus, an ...
-dependent overexpression of
BMI1
Polycomb complex protein BMI-1 also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51) is a protein that in humans is encoded by the ''BMI1'' gene (B cell-specific Moloney murine leukemia virus integration ...
and
BCL-XL
B-cell lymphoma-extra large (Bcl-xL), encoded by the BCL2-like 1 gene, is a transmembrane molecule in the mitochondria. It is a member of the Bcl-2 family of proteins, and acts as an anti-apoptotic protein by preventing the release of mitochondr ...
. The resulting imMKCLs can be expanded in culture over extended periods (4–5 months), even after
cryopreservation
Cryo-preservation or cryo-conservation is a process where Organism, organisms, organelles, cell (biology), cells, Biological tissue, tissues, extracellular matrix, Organ (anatomy), organs, or any other biological constructs susceptible to damage ...
. Halting the overexpression (by the removal of doxycycline from the medium) of c-MYC,
BMI1
Polycomb complex protein BMI-1 also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51) is a protein that in humans is encoded by the ''BMI1'' gene (B cell-specific Moloney murine leukemia virus integration ...
and
BCL-XL
B-cell lymphoma-extra large (Bcl-xL), encoded by the BCL2-like 1 gene, is a transmembrane molecule in the mitochondria. It is a member of the Bcl-2 family of proteins, and acts as an anti-apoptotic protein by preventing the release of mitochondr ...
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
GATA-binding factor 1 or GATA-1 (also termed Erythroid transcription factor) is the founding member of the GATA family of transcription factors. This protein is widely expressed throughout vertebrate species. In humans and mice, it is encoded by ...
,
FLI1
Friend leukemia integration 1 transcription factor (FLI1), also known as transcription factor ERGB, is a protein that in humans is encoded by the ''FLI1'' gene, which is a proto-oncogene.
Function
Fli-1 is a member of the ETS transcription fa ...
and
TAL1
__NOTOC__
T-cell acute lymphocytic leukemia protein 1 (i.e. TAL1 but also termed stem cell leukemia/T-cell acute leukemia 1 .e. SCL/TAL1 is a protein that in humans is encoded by the ''TAL1'' gene.
The protein encoded by TAL1 is a basic helix-lo ...
. The forward programmed
megakaryocyte
A megakaryocyte (''mega-'' + '' karyo-'' + '' -cyte'', "large-nucleus cell") is a large bone marrow cell with a lobated nucleus responsible for the production of blood thrombocytes (platelets), which are necessary for normal blood clotting. In hum ...
s proliferate and differentiate in culture for several months with megakaryocyte purity over 90% reaching up to 2x10
5 mature megakaryocytes per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as one million starting hPSCs.
See also overview
Immune cells
A specialised type of
white blood cell
White blood cells, also called leukocytes or leucocytes, are the cell (biology), cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and de ...
, known as
cytotoxic T lymphocytes
A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adap ...
(CTLs), are produced by the
immune system
The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathogens, from viruses to parasitic worms, as well as cancer cells and objects such as wood splinte ...
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
In biology, chimeric antigen receptors (CARs)—also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors—are receptor proteins that have been engineered to give T cells the new ability to target a specific ...
(CAR)
technologies to generate human T cells targeted to
CD19, an antigen expressed by malignant
B cell
B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or ...
s, 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
adjuvant In pharmacology, an adjuvant is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. Specifically, the term can refer to:
* Adjuvant therapy in cancer management
* Analgesic ...
s for cancer immunotherapy. iNKT cells act as innate T lymphocytes and serve as a bridge between the