![Human induced pluripotent stem cell colony (51816035910)](https://upload.wikimedia.org/wikipedia/commons/3/3e/Human_induced_pluripotent_stem_cell_colony_%2851816035910%29.jpg)
Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of
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 ...
stem cell
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 ...
that can be generated directly from a
somatic cell
A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells compo ...
. The iPSC technology was pioneered by
Shinya Yamanaka
is a Japanese stem cell researcher and a Nobel Prize laureate. He serves as the director of Center for iPS Cell (induced Pluripotent Stem Cell) Research and Application and a professor at the Institute for Frontier Medical Sciences at Kyo ...
's lab in
Kyoto
Kyoto (; Japanese: , ''Kyōto'' ), officially , is the capital city of Kyoto Prefecture in Japan. Located in the Kansai region on the island of Honshu, Kyoto forms a part of the Keihanshin metropolitan area along with Osaka and Kobe. , the ci ...
,
Japan
Japan ( ja, 日本, or , and formally , ''Nihonkoku'') is an island country in East Asia. It is situated in the northwest Pacific Ocean, and is bordered on the west by the Sea of Japan, while extending from the Sea of Okhotsk in the north ...
, who showed in 2006 that the introduction of four specific genes (named
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 refe ...
,
Oct3/4
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 and
Klf4), collectively known as Yamanaka factors, encoding
transcription factors
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 func ...
could convert somatic cells into pluripotent stem cells.
He was awarded the 2012 Nobel Prize along with Sir
John Gurdon
Sir John Bertrand Gurdon (born 2 October 1933) is a British developmental biologist. He is best known for his pioneering research in nuclear transplantation and cloning. He was awarded the Lasker Award in 2009. In 2012, he and Shinya Yamanaka ...
"for the discovery that mature cells can be reprogrammed to become pluripotent."
Pluripotent stem cells hold promise in the field of
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 ...
.
Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.
The most well-known type of pluripotent stem cell is the
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 ...
. However, since the generation of embryonic stem cells involves destruction (or at least manipulation)
of the pre-implantation stage embryo, there has been much
controversy
Controversy is a state of prolonged public dispute or debate, usually concerning a matter of conflicting opinion or point of view. The word was coined from the Latin ''controversia'', as a composite of ''controversus'' – "turned in an opposite d ...
surrounding their use. Patient-matched embryonic stem cell lines can now be derived using
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 implanting a ...
(SCNT).
Since iPSCs can be derived directly from adult tissues, they not only bypass the need for embryos, but can be made in a patient-matched manner, which means that each individual could have their own pluripotent stem cell line. These unlimited supplies of
autologous
Autotransplantation is the transplantation of organs, tissues, or even particular proteins from one part of the body to another in the same person ('' auto-'' meaning "self" in Greek).
The autologous tissue (also called autogenous, autogene ...
cells could be used to generate transplants without the risk of immune rejection. While the iPSC technology has not yet advanced to a stage where therapeutic transplants have been deemed safe, iPSCs are readily being used in personalized drug discovery efforts and understanding the patient-specific basis of disease.
Yamanaka named iPSCs with a lower case "i" due to the popularity of the
iPod
The iPod is a discontinued series of portable media players and multi-purpose mobile devices designed and marketed by Apple Inc. The first version was released on October 23, 2001, about months after the Macintosh version of iTunes ...
and other products.
In his Nobel seminar, Yamanaka cited the earlier seminal work of
Harold Weintraub
Harold M. "Hal" Weintraub was an American scientist who lived from 1945 until his death in 1995 from an aggressive brain tumor. Only 49 years old, Weintraub left behind a legacy of research.
Early life and education
Born on June 2, 1945, in ...
on the role of
myoblast determination protein 1 (MyoD) in reprogramming cell fate to a muscle lineage as an important precursor to the discovery of iPSCs.
Production
iPSCs are typically derived by introducing products of specific sets of pluripotency-associated genes, or "reprogramming factors", into a given cell type. The original set of reprogramming factors (also dubbed Yamanaka factors) are the transcription factors
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 ...
(Pou5f1),
Sox2,
Klf4 and
cMyc. While this combination is most conventional in producing iPSCs, each of the factors can be functionally replaced by related transcription factors,
miRNAs
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miR ...
, small molecules, or even non-related genes such as lineage specifiers.
It is also clear that pro-mitotic factors such as C-MYC/L-MYC or repression of cell cycle checkpoints, such as p53, are conduits to creating a compliant cellular state for iPSC reprograming .
iPSC derivation is typically a slow and inefficient process, taking 1–2 weeks for mouse cells and 3–4 weeks for human cells, with efficiencies around 0.01–0.1%. However, considerable advances have been made in improving the efficiency and the time it takes to obtain iPSCs. Upon introduction of reprogramming factors, cells begin to form colonies that resemble pluripotent stem cells, which can be isolated based on their morphology, conditions that select for their growth, or through expression of surface markers or
reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
s.
First generation (mouse)
Induced pluripotent stem cells were first generated by
Shinya Yamanaka
is a Japanese stem cell researcher and a Nobel Prize laureate. He serves as the director of Center for iPS Cell (induced Pluripotent Stem Cell) Research and Application and a professor at the Institute for Frontier Medical Sciences at Kyo ...
's team at
Kyoto University
, mottoeng = Freedom of academic culture
, established =
, type = National university, Public (National)
, endowment = ¥ 316 billion (2.4 1000000000 (number), billion USD)
, faculty = 3,480 (Teaching Staff)
, administrative_staff ...
, Japan, in 2006.
They hypothesized that genes important to embryonic stem cell (ESC) function might be able to induce an embryonic state in adult cells. They chose twenty-four genes previously identified as important in ESCs and used retroviruses to deliver these genes to mouse
fibroblasts
A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework ( stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells o ...
. The fibroblasts were engineered so that any cells reactivating the ESC-specific gene,
Fbx15
F-box protein 15 also known as Fbx15 is a protein that in humans is encoded by the FBXO15 gene.
Function
Members of the F-box protein family, such as FBXO15, are characterized by an approximately 40-amino acid F-box motif. SCF complexes, formed ...
, could be isolated using antibiotic selection.
Upon delivery of all twenty-four factors, ESC-like colonies emerged that reactivated the Fbx15 reporter and could propagate indefinitely. To identify the genes necessary for reprogramming, the researchers removed one factor at a time from the pool of twenty-four. By this process, they identified four factors, Oct4, Sox2, cMyc, and Klf4, which were each necessary and together sufficient to generate ESC-like colonies under selection for reactivation of Fbx15.
Second generation (mouse)
In June 2007, three separate research groups, including that of Yamanaka's, a
Harvard
Harvard University is a private Ivy League research university in Cambridge, Massachusetts. Founded in 1636 as Harvard College and named for its first benefactor, the Puritan clergyman John Harvard, it is the oldest institution of higher le ...
/
University of California, Los Angeles
The University of California, Los Angeles (UCLA) is a public land-grant research university in Los Angeles, California. UCLA's academic roots were established in 1881 as a teachers college then known as the southern branch of the California St ...
collaboration, and a group at
MIT
The Massachusetts Institute of Technology (MIT) is a private land-grant research university in Cambridge, Massachusetts. Established in 1861, MIT has played a key role in the development of modern technology and science, and is one of the m ...
, published studies that substantially improved on the reprogramming approach, giving rise to iPSCs that were indistinguishable from ESCs. Unlike the first generation of iPSCs, these second generation iPSCs produced viable chimeric mice and contributed to the mouse germline, thereby achieving the 'gold standard' for pluripotent stem cells.
These second-generation iPSCs were derived from mouse fibroblasts by retroviral-mediated expression of the same four transcription factors (Oct4, Sox2, cMyc, Klf4). However, instead of using Fbx15 to select for pluripotent cells, the researchers used
Nanog, a gene that is functionally important in ESCs. By using this different strategy, the researchers created iPSCs that were functionally identical to ESCs.
Human induced pluripotent stem cells
Generation from human fibroblasts
Reprogramming of human cells to iPSCs was reported in November 2007 by two independent research groups:
Shinya Yamanaka
is a Japanese stem cell researcher and a Nobel Prize laureate. He serves as the director of Center for iPS Cell (induced Pluripotent Stem Cell) Research and Application and a professor at the Institute for Frontier Medical Sciences at Kyo ...
of Kyoto University, Japan, who pioneered the original iPSC method, and
James Thomson of
University of Wisconsin-Madison
A university () is an institution of higher (or tertiary) education and research which awards academic degrees in several academic disciplines. Universities typically offer both undergraduate and postgraduate programs. In the United States, the ...
who was the first to derive human embryonic stem cells. With the same principle used in mouse reprogramming, Yamanaka's group successfully transformed human fibroblasts into iPSCs with the same four pivotal genes, Oct4, Sox2, Klf4, and cMyc, using a
retroviral
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 transcriptas ...
system,
while Thomson and colleagues used a different set of factors, Oct4, Sox2, Nanog, and Lin28, using a
lentiviral
''Lentivirus'' is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lent ...
system.
Generation from additional cell types
Obtaining fibroblasts to produce iPSCs involves a skin biopsy, and there has been a push towards identifying cell types that are more easily accessible.
In 2008, iPSCs were derived from human keratinocytes, which could be obtained from a single hair pluck.
In 2010, iPSCs were derived from peripheral blood cells,
and in 2012, iPSCs were made from renal epithelial cells in the urine.
Other considerations for starting cell type include mutational load (for example, skin cells may harbor more mutations due to UV exposure),
time it takes to expand the population of starting cells,
and the ability to differentiate into a given cell type.
Genes used to produce iPSCs
The generation of induced pluripotent cells is crucially dependent on the transcription factors used for the induction.
Oct-3/4 and certain products of the
Sox gene family
''SOX'' genes ('' SRY''-related HMG-box genes) encode a family of transcription factors that bind to the minor groove in DNA, and belong to a super-family of genes characterized by a homologous sequence called the HMG-box (for high mobility g ...
(Sox1, Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the
Klf family (Klf1, Klf2, Klf4, and Klf5), the
Myc family (c-myc, L-myc, and N-myc),
Nanog, and
LIN28
Lin-28 homolog A is a protein that in humans is encoded by the ''LIN28'' gene.
LIN28 encodes an RNA-binding protein that binds to and enhances the translation of the IGF-2 (insulin-like growth factor 2) mRNA. Lin28 binds to the let-7 pre-microRN ...
, have been identified to increase the induction efficiency.
*
Oct-3/4 (Pou5f1) Oct-3/4 is one of the family of
octamer ("Oct") transcription factors, and plays a crucial role in maintaining pluripotency. The absence of Oct-3/4 in Oct-3/4
+ cells, such as
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 embryonic stem cells, leads to spontaneous
trophoblast
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 p ...
differentiation, and presence of Oct-3/4 thus gives rise to the pluripotency and differentiation potential of embryonic stem cells. Various other genes in the "Oct" family, including Oct-3/4's close relatives,
Oct1 and
Oct6
POU domain, class 3, transcription factor 1 (also known as Oct-6) is a protein that in humans is encoded by the ''POU3F1'' gene.
See also
* Octamer transcription factor
Octamer transcription factors are a protein family, family of transcriptio ...
, fail to elicit induction, thus demonstrating the exclusiveness of Oct-3/4 to the induction process. However a team headed by Hans Schöler (who discovered the Oct4 gene back in 1989) showed that Oct4 overexpression during reprogramming causes epigenetic changes deteriorating the quality of iPSCs. Comparing to OSKM (Oct4, Sox2, Klf4 and c-Myc) new SKM (Sox2, Klf4 and c-Myc) reprogramming generates iPSCs with developmental potential equivalent to embryonic stem cell, as determined by their ability to generate all-iPSC mice through
tetraploid embryo complementation.
*
Sox family
Sox most often refers to:
* Boston Red Sox, an MLB team
* Chicago White Sox, an MLB team
* An alternate spelling of socks
Sox may also refer to:
Places
* SOX, Sogamoso Airport's IATA airport code, an airport in Colombia
Computing and technolo ...
: The Sox family of transcription factors is associated with maintaining pluripotency similar to Oct-3/4, although it is associated with multipotent and unipotent stem cells in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem cells. While
Sox2 was the initial gene used for induction by Yamanaka et al., Jaenisch et al., and Thomson et al., other transcription factors in the Sox family have been found to work as well in the induction process.
Sox1
SOX1 is a gene that encodes a transcription factor with a HMG-box (high mobility group) DNA-binding domain and functions primarily in neurogenesis. SOX1, SOX2 and SOX3, members of the SOX gene family (specifically the SOXB1 group), contain tra ...
yields iPS cells with a similar efficiency as Sox2, and genes
Sox3
Transcription factor SOX-3 is a protein that in humans is encoded by the ''SOX3'' gene. This gene encodes a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the regulation of embryonic brain development and in de ...
,
Sox15, and
Sox18
Transcription factor SOX-18 is a protein that in humans is encoded by the ''SOX18'' gene.
Function
This gene encodes a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the regulation of embryonic development ...
also generate iPS cells, although with decreased efficiency.
*
Klf family:
Klf4 of the Klf family of transcription factors was initially identified by Yamanaka et al. and confirmed by Jaenisch et al. As a factor for the generation of mouse iPS cells and was demonstrated by Yamanaka et al. as a factor for generation of human iPS cells. However, Thomson et al. reported that Klf4 was unnecessary for generation of human iPS cells and in fact failed to generate human iPS cells.
Klf2
Krüppel-like Factor 2 (KLF2), also known as lung Krüppel-like Factor (LKLF), is a protein that in humans is encoded by the ''KLF2'' gene on chromosome 19. It is in the Krüppel-like factor family of zinc finger transcription factors, and it has ...
and Klf4 were found to be factors capable of generating iPS cells, and related genes
Klf1 and
Klf5
Krueppel-like factor 5 is a protein that in humans is encoded by the ''KLF5'' gene.
Function
This gene encodes a member of the Kruppel-like factor subfamily of zinc finger proteins. Since the protein localizes to the nucleus and binds the epid ...
did as well, although with reduced efficiency.
*
Myc family: The Myc family of transcription factors are
proto-oncogenes
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. implicated in cancer. Yamanaka et al. and Jaenisch et al. demonstrated that c-myc is a factor implicated in the generation of mouse iPS cells and Yamanaka et al. demonstrated it was a factor implicated in the generation of human iPS cells. However, Thomson et al., Yamanaka et al. usage of the "myc" family of genes in induction of iPS cells is troubling for the eventuality of iPS cells as clinical therapies, as 25% of mice transplanted with c-myc-induced iPS cells developed lethal
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.
N-myc
N-myc proto-oncogene protein also known as N-Myc or basic helix-loop-helix protein 37 (bHLHe37), is a protein that in humans is encoded by the ''MYCN'' gene.
Function
The ''MYCN'' gene is a member of the MYC family of transcription factors an ...
and
L-myc have been identified to induce instead of c-myc with similar efficiency.
*
Nanog: In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, is necessary in promoting pluripotency. Therefore, it was surprising when Yamanaka et al. reported that Nanog was unnecessary for induction although Thomson et al. has reported it is possible to generate iPS cells with Nanog as one of the factors.
*
LIN28
Lin-28 homolog A is a protein that in humans is encoded by the ''LIN28'' gene.
LIN28 encodes an RNA-binding protein that binds to and enhances the translation of the IGF-2 (insulin-like growth factor 2) mRNA. Lin28 binds to the let-7 pre-microRN ...
: LIN28 is an
mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells associated with differentiation and proliferation. Thomson et al. demonstrated that
LIN28
Lin-28 homolog A is a protein that in humans is encoded by the ''LIN28'' gene.
LIN28 encodes an RNA-binding protein that binds to and enhances the translation of the IGF-2 (insulin-like growth factor 2) mRNA. Lin28 binds to the let-7 pre-microRN ...
is a factor in iPSC generation in combination with OCT4, SOX2, and NANOG.
*
Glis1: Glis1 is transcription factor that can be used with Oct-3/4, Sox2 and Klf4 to induce pluripotency. It poses numerous advantages when used instead of C-myc.
Challenges in reprogramming cells to pluripotency
Although the methods pioneered by Yamanaka and others have demonstrated that adult cells can be reprogrammed to iPS cells, there are still challenges associated with this technology:
# Low efficiency: in general, the conversion to iPS cells has been incredibly low. For example, the rate at which
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 ...
cells were reprogrammed into iPS cells in Yamanaka's original mouse study was 0.01–0.1%.
The low efficiency rate may reflect the need for precise timing, balance, and absolute levels of expression of the reprogramming genes. It may also suggest a need for rare genetic or epigenetic changes in the original
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 ...
cell population or in the prolonged culture. However, recently a path was found for efficient reprogramming which required downregulation of the
nucleosome
A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamen ...
remodeling and deacetylation (
NuRD
In the field of molecular biology, the Mi-2/NuRD (Nucleosome Remodeling Deacetylase) complex, is a group of associated proteins with both ATP-dependent chromatin remodeling and histone deacetylase activities. , Mi-2/NuRD was the only known prot ...
) complex. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs. Depletion of Mbd3, on the other hand, improves reprogramming efficiency,
that results in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells).
# Genomic Insertion: genomic integration of the transcription factors limits the utility of the transcription factor approach because of the risk of mutations being inserted into the target cell's genome.
A common strategy for avoiding genomic insertion has been to use a different vector for input.
Plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s,
adenovirus
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
es, and
transposon
A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transpo ...
vectors have all been explored, but these often come with the tradeoff of lower throughput.
# Tumorigenicity: Depending on the methods used, reprogramming of adult cells to obtain iPSCs may pose significant risks that could limit their use in humans. For example, if viruses are used to genomically alter the cells, the expression of
oncogenes
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. (cancer-causing genes) may potentially be triggered. In February 2008, scientists announced the discovery of a technique that could remove oncogenes after the induction of pluripotency, thereby increasing the potential use of iPS cells in human diseases.
In another study, Yamanaka reported that one can create iPSCs without the oncogene c-Myc. The process took longer and was not as efficient, but the resulting chimeras did not develop cancer.
Inactivation or deletion of the tumor suppressor p53, which is a key regulator of cancer, significantly increases reprogramming efficiency.
Thus there seems to be a tradeoff between reprogramming efficiency and tumor generation.
#Incomplete reprogramming: reprogramming also faces the challenge of completeness. This is particularly challenging because the genome-wide
epigenetic code must be reformatted to that of the target cell type in order to fully reprogram a cell. However, three separate groups were able to find 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 ...
(MEF)-derived iPS cells that could be injected into
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 ...
blastocysts
The blastocyst is a structure formed in the early embryonic development of mammals. It possesses an inner cell mass (ICM) also known as the ''embryoblast'' which subsequently forms the embryo, and an outer layer of trophoblast cells called the t ...
and resulted in the live birth of mice derived entirely from iPS cells, thus ending the debate over the equivalence of embryonic stem cells (ESCs) and iPS with regard to pluripotency.
The table on the right summarizes the key strategies and techniques used to develop iPS cells in the first five years after Yamanaka et al.'s 2006 breakthrough. Rows of similar colors represent studies that used similar strategies for reprogramming.
Alternative approaches
Mimicking transcription factors with chemicals
One of the main strategies for avoiding problems (1) and (2) has been to use
small molecule
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 ar ...
s that can mimic the effects of transcription factors. These compounds can compensate for a reprogramming factor that does not effectively target the genome or fails at reprogramming for another reason; thus they raise reprogramming efficiency. They also avoid the problem of genomic integration, which in some cases contributes to tumor genesis. Key studies using such strategy were conducted in 2008. Melton et al. studied the effects of
histone deacetylase (HDAC) inhibitor valproic acid. They found that it increased reprogramming efficiency 100-fold (compared to Yamanaka's traditional transcription factor method).
The researchers proposed that this compound was mimicking the signaling that is usually caused by the transcription factor c-Myc. A similar type of compensation mechanism was proposed to mimic the effects of
Sox2. In 2008, Ding et al. used the inhibition of histone methyl transferase (HMT) with BIX-01294 in combination with the activation of
calcium channels
A calcium channel is an ion channel which shows selective permeability to calcium ions. It is sometimes synonymous with voltage-gated calcium channel, although there are also ligand-gated calcium channels.
Comparison tables
The following tables ex ...
in the plasma membrane in order to increase reprogramming efficiency.
Deng et al. of Beijing University reported in July 2013 that induced pluripotent stem cells can be created without any genetic modification. They used a cocktail of seven small-molecule compounds including DZNep to induce the mouse somatic cells into stem cells which they called CiPS cells with the efficiency – at 0.2% – comparable to those using standard iPSC production techniques. The CiPS cells were introduced into developing mouse embryos and were found to contribute to all major cells types, proving its pluripotency.
Ding et al. demonstrated an alternative to transcription factor reprogramming through the use of drug-like chemicals. By studying the MET (
mesenchymal-epithelial transition) process in which fibroblasts are pushed to a stem-cell like state, Ding's group identified two chemicals – ALK5 inhibitor SB431412 and MEK (mitogen-activated protein kinase) inhibitor PD0325901 – which was found to increase the efficiency of the classical genetic method by 100 fold. Adding a third compound known to be involved in the cell survival pathway,
Thiazovivin further increases the efficiency by 200 fold. Using the combination of these three compounds also decreased the reprogramming process of the human fibroblasts from four weeks to two weeks.
In April 2009, it was demonstrated that generation of iPS cells is possible without any genetic alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled into the cells via
poly-arginine anchors was sufficient to induce pluripotency.
The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells).
Alternate vectors
Another key strategy for avoiding problems such as tumorgenesis and low throughput has been to use alternate forms of vectors:
adenovirus
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
,
plasmids
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
, and naked DNA or protein compounds.
In 2008, Hochedlinger et al. used an
adenovirus
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
to transport the requisite four transcription factors into the DNA of skin and liver cells of mice, resulting in cells identical to ESCs. The
adenovirus
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
is unique from other vectors like viruses and retroviruses because it does not incorporate any of its own genes into the targeted host and avoids the potential for insertional mutagenesis.
In 2009, Freed et al. demonstrated successful reprogramming of human fibroblasts to iPS cells. Another advantage of using
adenoviruses
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
is that they only need to present for a brief amount of time in order for effective reprogramming to take place.
Also in 2008, Yamanaka et al. found that they could transfer the four necessary genes with a plasmid.
The Yamanaka group successfully reprogrammed mouse cells by transfection with two plasmid constructs carrying the reprogramming factors; the first plasmid expressed c-Myc, while the second expressed the other three factors (
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 ...
,
Klf4, and
Sox2). Although the plasmid methods avoid viruses, they still require cancer-promoting genes to accomplish reprogramming. The other main issue with these methods is that they tend to be much less efficient compared to retroviral methods. Furthermore, transfected
plasmids
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
have been shown to integrate into the host genome and therefore they still pose the risk of insertional mutagenesis. Because non-retroviral approaches have demonstrated such low efficiency levels, researchers have attempted to effectively rescue the technique with what is known as the
PiggyBac Transposon System. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving footprint mutations in the host cell genome. The PiggyBac Transposon System involves the re-excision of exogenous genes, which eliminates the issue of insertional mutagenesis.
Stimulus-triggered acquisition of pluripotency cell
In January 2014, two articles were published claiming that a type of pluripotent stem cell can be generated by subjecting the cells to certain types of stress (bacterial toxin, a low pH of 5.7, or physical squeezing); the resulting cells were called STAP cells, for
stimulus-triggered acquisition of pluripotency.
In light of difficulties that other labs had replicating the results of the surprising study, in March 2014, one of the co-authors has called for the articles to be retracted. On 4 June 2014, the lead author,
Obokata agreed to retract both the papers
after she was found to have committed 'research misconduct' as concluded in an investigation by
RIKEN on 1 April 2014.
RNA molecules
MicroRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
s are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Measuring variations in microRNA expression in iPS cells can be used to predict their differentiation potential.
Addition of microRNAs can also be used to enhance iPS potential. Several mechanisms have been proposed.
ES cell-specific
microRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
molecules (such as miR-291, miR-294 and miR-295) enhance the efficiency of induced pluripotency by acting downstream of c-Myc.
microRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
s can also block expression of repressors of Yamanaka's four transcription factors, and there may be additional mechanisms induce reprogramming even in the absence of added exogenous transcription factors.
Identity
![Three germ line cells differentiated from iPSCs](https://upload.wikimedia.org/wikipedia/commons/7/75/Three_germ_line_cells_differentiated_from_iPSCs.png)
Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem cells, in many aspects, such as the expression of certain stem cell genes and proteins,
chromatin methylation patterns, doubling time,
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 ...
formation,
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 ...
formation, viable
chimera
Chimera, Chimaera, or Chimaira (Greek for " she-goat") originally referred to:
* Chimera (mythology), a fire-breathing monster of Ancient Lycia said to combine parts from multiple animals
* Mount Chimaera, a fire-spewing region of Lycia or Cilicia ...
formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed.
Gene expression and genome-wide
H3K4me3
H3K4me3 is an epigenetic modification to the DNA packaging protein Histone H3 that indicates tri-methylation at the 4th lysine residue of the histone H3 protein and is often involved in the regulation of gene expression. The name denotes the add ...
and
H3K27me3
H3K27me3 is an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation of lysine 27 on histone H3 protein.
This tri-methylation is associated with the downregulation of nearby genes via t ...
were found to be extremely similar between ES and iPS cells. The generated iPSCs were remarkably similar to naturally isolated pluripotent stem cells (such as mouse and human embryonic stem cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity, authenticity, and pluripotency of iPSCs to naturally isolated pluripotent stem cells:
*Cellular biological properties
**Morphology: iPSCs were morphologically similar to ESCs. Each cell had round shape, large
nucleolus
The nucleolus (, plural: nucleoli ) is the largest structure in the nucleus of eukaryotic cells. It is best known as the site of ribosome biogenesis, which is the synthesis of ribosomes. The nucleolus also participates in the formation of sig ...
and scant
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
. Colonies of iPSCs were also similar to that of ESCs. Human iPSCs formed sharp-edged, flat, tightly packed colonies similar to hESCs and mouse iPSCs formed the colonies similar to mESCs, less flat and more aggregated colonies than that of hESCs.
**Growth properties: Doubling time and
mitotic
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is maintai ...
activity are cornerstones of ESCs, as stem cells must self-renew as part of their definition. iPSCs were mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs.
**Stem cell markers: iPSCs expressed cell surface antigenic markers expressed on ESCs. Human iPSCs expressed the markers specific to hESC, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPSCs expressed SSEA-1 but not SSEA-3 nor SSEA-4, similarly to mESCs.
**Stem Cell Genes: iPSCs expressed genes expressed in undifferentiated ESCs, including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.
**Telomerase activity:
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 ...
s are necessary to sustain cell division unrestricted by the
Hayflick limit of ~50 cell divisions. hESCs express high telomerase activity to sustain self-renewal and proliferation, and iPSCs also demonstrate high telomerase activity and express hTERT (human
telomerase reverse transcriptase
Telomerase reverse transcriptase (abbreviated to TERT, or hTERT in humans) is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex.
T ...
), a necessary component in the telomerase protein complex.
*Pluripotency: iPSCs were capable of differentiation in a fashion similar to ESCs into fully differentiated tissues.
**Neural differentiation: iPSCs were differentiated into
neuron
A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...
s, expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence of
catecholamine
A catecholamine (; abbreviated CA) is a monoamine neurotransmitter, an organic compound that has a catechol (benzene with two hydroxyl side groups next to each other) and a side-chain amine.
Catechol can be either a free molecule or a subst ...
-associated enzymes may indicate that iPSCs, like hESCs, may be differentiable into
dopaminergic
Dopaminergic means "related to dopamine" (literally, "working on dopamine"), dopamine being a common neurotransmitter. Dopaminergic substances or actions increase dopamine-related activity in the brain. Dopaminergic brain pathways facilitate d ...
neurons. Stem cell-associated genes were downregulated after differentiation.
**Cardiac differentiation: iPSCs were differentiated into
cardiomyocytes
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 tha ...
that spontaneously began beating. Cardiomyocytes expressed TnTc, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-associated genes were downregulated after differentiation.
**Teratoma formation: iPSCs injected into
immunodeficient
Immunodeficiency, also known as immunocompromisation, is a state in which the immune system's ability to fight infectious diseases and cancer is compromised or entirely absent. Most cases are acquired ("secondary") due to extrinsic factors that a ...
mice spontaneously formed
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 after nine weeks. Teratomas are tumors of multiple lineages containing tissue derived from the 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 pr ...
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 ...
,
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 ...
and
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 ...
; this is unlike other tumors, which typically are of only one cell type. Teratoma formation is a landmark test for pluripotency.
**Embryoid body: hESCs in culture spontaneously form ball-like embryo-like structures termed "
embryoid bodies", which consist of a core of mitotically active and differentiating hESCs and a periphery of fully differentiated cells from all three germ layers. iPSCs also form embryoid bodies and have peripheral differentiated cells.
**Chimeric mice: hESCs naturally reside within the inner cell mass (
embryoblast
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 t ...
) of
blastocyst
The blastocyst is a structure formed in the early embryonic development of mammals. It possesses an inner cell mass (ICM) also known as the ''embryoblast'' which subsequently forms the embryo, and an outer layer of trophoblast cells called the t ...
s, and in the embryoblast, differentiate into the embryo while the blastocyst's shell (
trophoblast
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 p ...
) differentiates into extraembryonic tissues. The hollow trophoblast is unable to form a living embryo, and thus it is necessary for the embryonic stem cells within the embryoblast to differentiate and form the embryo. iPSCs were injected by
micropipette
A pipette (sometimes spelled as pipett) is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser. Pipettes come in several designs for various purposes with diff ...
into a trophoblast, and the blastocyst was transferred to recipient females.
Chimeric living mouse pups were created: mice with iPSC derivatives incorporated all across their bodies with 10–90% chimerism.
**
Tetraploid complementation: iPS cells from mouse fetal fibroblasts injected into tetraploid blastocysts (which themselves can only form extra-embryonic tissues) can form whole, non-chimeric, fertile mice, although with low success rate.
*Epigenetic reprogramming
**Promoter demethylation: Methylation is the transfer of a methyl group to a DNA base, typically the transfer of a methyl group to a cytosine molecule in a CpG site (adjacent cytosine/guanine sequence). Widespread methylation of a gene interferes with
expression
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, o ...
by preventing the activity of expression proteins, or by recruiting enzymes that interfere with expression. Thus, methylation of a gene effectively silences it by preventing transcription. Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in iPSCs, demonstrating their promoter activity and the active promotion and expression of pluripotency-associated genes in iPSCs.
**DNA methylation globally: Human iPS cells are highly similar to ES cells in their patterns of which
cytosine
Cytosine () ( symbol C or Cyt) is one of the four nucleobases found in DNA and RNA, along with adenine, guanine, and thymine (uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached (an am ...
s are
methylated
In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These ...
, more than to any other cell type. However, on the order of a thousand sites show differences in several iPS cell lines. Half of these resemble the somatic cell line the iPS cells were derived from, the rest are iPSC-specific. Tens of regions which are
megabase
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s in size have also been found where iPS cells are not reprogrammed to the ES cell state.
**Histone demethylation:
Histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn a ...
s are compacting proteins that are structurally localized to DNA sequences that can affect their activity through various chromatin-related modifications. H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated, indicating the expression of Oct-3/4, Sox2, and Nanog.
Safety
*The major concern with the potential clinical application of iPSCs is their propensity to form tumors. Much the same as ESC, iPSCs readily form
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 ...
when injected into immunodeficient mice. Teratoma formation is considered a major obstacle to stem-cell based regenerative medicine by the FDA.
*A more recent study on motor functional recovery after spinal cord injuries in mice showed that after human-induced pluripotent stem cells were transplanted into the mice, the cells differentiated into three neural lineages in the spinal cord. The cells stimulated regrowth of the damaged spinal cord, maintained myelination, and formed synapses. These positive outcomes were observed for over 112 days after the spinal cord injury, without tumor formation. Nevertheless, a follow-up study by the same group showed distinct clones of human-induced pluripotent stem cells eventually formed tumors.
*Since iPSCs can only be produced with high efficiency at this time using modifications, they are generally predicted to be less safe and more tumorigenic than hESC. All the genes that have been shown to promote iPSC formation have also been linked to cancer in one way or another. Some of the genes are known oncogenes, including the members of the Myc family. While omitting Myc still allows for IPSC formation, the efficiency is reduced up to 100 fold.
*A non-genetic method of producing iPSCs has been demonstrated using recombinant proteins, but its efficiency was quite low.
However, refinements to this methodology yielding higher efficiency may lead to production of safer iPSCs. Other approaches such as using adenovirus or plasmids are generally thought to be safer than retroviral methods.
*An important area for future studies in the iPSC field is directly testing iPSC tumorigenicity using methods that mimic the approaches that would be used for regenerative medicine therapies. Such studies are crucial since iPSCs not only form teratoma, but also mice derived from iPSCs have a high incidence of death from malignant cancer.
A 2010 paper was published in the journal Stem Cells indicating that iPS cells are far more tumorigenic than ESC, supporting the notion that iPS cell safety is a serious concern.
*Concern regarding the immunogenicity of IPS cells arose in 2011 when Zhou et al. performed a study involving a teratoma formation assay and demonstrated that IPS cells produced an immune response strong enough to cause rejection of the cells. When a similar procedure was performed on genetically equivalent ES cells however, Zhou et al. found
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 indicated that the cells were tolerated by the immune system.
In 2013, Araki et al. attempted to reproduce the conclusion obtained by Zhou et al. using a different procedure. They took cells from a chimera that had been grown from IPSC clones and a mouse embryo, this tissue was then transplanted into
syngenic
The word "syngenic" or "syngeneic" (from the Greek word for a relative) means genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation. For example, it may be used for something transplanted fr ...
mice. They conducted a similar trial using ES cells instead of IPSC clone and compared the results. Findings indicate that there was no significant difference in the immunogenic response produced by the IPS cells and the ES cells. Furthermore, Araki et al. reported little or no immunogenic response for both cell lines.
Thus, Araki et al. was unable to come to the same conclusion as Zhou et al.
Recent achievements and future tasks for safe iPSC-based cell therapy are collected in the review of Okano et al.
Medical research
The task of producing iPS cells continues to be challenging due to the six problems mentioned above. A key tradeoff to overcome is that between efficiency and genomic integration. Most methods that do not rely on the integration of transgenes are inefficient, while those that do rely on the integration of transgenes face the problems of incomplete reprogramming and tumor genesis, although a vast number of techniques and methods have been attempted. Another large set of strategies is to perform a proteomic characterization of iPS cells.
Further studies and new strategies should generate optimal solutions to the five main challenges. One approach might attempt to combine the positive attributes of these strategies into an ultimately effective technique for reprogramming cells to iPS cells.
Another approach is the use of iPS cells derived from patients to identify therapeutic drugs able to rescue a phenotype. For instance, iPS cell lines derived from patients affected by ectodermal dysplasia syndrome (EEC), in which the
p63 gene is mutated, display abnormal epithelial commitment that could be partially rescued by a small compound.
Disease modeling and drug development
An attractive feature of human iPS cells is the ability to derive them from adult patients to study the cellular basis of human disease. Since iPS cells are self-renewing and pluripotent, they represent a theoretically unlimited source of patient-derived cells which can be turned into any type of cell in the body. This is particularly important because many other types of human cells derived from patients tend to stop growing after a few passages in laboratory culture. iPS cells have been generated for a wide variety of human genetic diseases, including common disorders such as Down syndrome and polycystic kidney disease.
In many instances, the patient-derived iPS cells exhibit cellular defects not observed in iPS cells from healthy subjects, providing insight into the pathophysiology of the disease.
An international collaborated project, StemBANCC, was formed in 2012 to build a collection of iPS cell lines for drug screening for a variety of disease. Managed by the
University of Oxford
, mottoeng = The Lord is my light
, established =
, endowment = £6.1 billion (including colleges) (2019)
, budget = £2.145 billion (2019–20)
, chancellor ...
, the effort pooled funds and resources from 10 pharmaceutical companies and 23 universities. The goal is to generate a library of 1,500 iPS cell lines which will be used in early drug testing by providing a simulated human disease environment. Furthermore, combining hiPSC technology and small molecule or genetically encoded voltage and calcium indicators provided a large-scale and high-throughput platform for cardiovascular drug safety screening.
Organ synthesis
A proof-of-concept of using induced pluripotent stem cells (iPSCs) to generate human organ for
transplantation was reported by researchers from Japan. Human '
liver
The liver is a major Organ (anatomy), organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of proteins and biochemicals necessary for ...
buds' (iPSC-LBs) were grown from a mixture of three different kinds of stem cells:
hepatocytes
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, b ...
(for liver function) coaxed from iPSCs;
endothelial stem cells (to form lining of
blood vessels
The blood vessels are the components of the circulatory system that transport blood throughout the human body. These vessels transport blood cells, nutrients, and oxygen to the tissues of the body. They also take waste and carbon dioxide away f ...
) from
umbilical cord blood
Cord blood (umbilical cord blood) is blood that remains in the placenta and in the attached umbilical cord after childbirth. Cord blood is collected because it contains stem cells, which can be used to treat hematopoietic and genetic disorders su ...
; and
mesenchymal stem cells (to form
connective tissue
Connective tissue is one of the four primary types of animal tissue, along with epithelial tissue, muscle tissue, and nervous tissue. It develops from the mesenchyme derived from the mesoderm the middle embryonic germ layer. Connective tiss ...
). This new approach allows different cell types to self-organize into a complex organ, mimicking the process in
fetal development
Prenatal development () includes the development of the embryo and of the fetus during a viviparous animal's gestation. Prenatal development starts with fertilization, in the germinal stage of embryonic development, and continues in fetal devel ...
. After growing ''in vitro'' for a few days, the liver buds were transplanted into mice where the 'liver' quickly connected with the host blood vessels and continued to grow. Most importantly, it performed regular liver functions including metabolizing drugs and producing liver-specific proteins. Further studies will monitor the longevity of the transplanted organ in the host body (ability to integrate or avoid
rejection) and whether it will transform into
tumors
A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
.
Using this method, cells from one mouse could be used to test 1,000 drug compounds to treat liver disease, and reduce animal use by up to 50,000.
Organ regeneration
In 2021, a switchable Yamanaka factors-
reprogramming
In biology, reprogramming refers to erasure and remodeling of epigenetic marks, such as DNA methylation, during mammalian development or in cell culture. Such control is also often associated with alternative covalent modifications of histones.
...
-based approach for
regeneration
Regeneration may refer to:
Science and technology
* Regeneration (biology), the ability to recreate lost or damaged cells, tissues, organs and limbs
* Regeneration (ecology), the ability of ecosystems to regenerate biomass, using photosynthesis
...
of damaged heart without tumor-formation was demonstrated in mice and was successful if the intervention was carried out immediately before or after a heart attack.
Tissue repair
Embryonic cord-blood cells were induced into pluripotent stem cells using plasmid DNA. Using cell surface endothelial/pericytic markers
CD31
Platelet endothelial cell adhesion molecule (PECAM-1) also known as cluster of differentiation 31 (CD31) is a protein that in humans is encoded by the ''PECAM1'' gene found on chromosome17q23.3. PECAM-1 plays a key role in removing aged neutrop ...
and
CD146, researchers identified 'vascular progenitor', the high-quality, multipotent vascular stem cells. After the iPS cells were injected directly into the
vitreous of the damaged
retina
The retina (from la, rete "net") is the innermost, light-sensitive layer of tissue of the eye of most vertebrates and some molluscs. The optics of the eye create a focused two-dimensional image of the visual world on the retina, which then ...
of mice, the stem cells engrafted into the retina, grew and repaired the
vascular vessels.
Labelled iPSCs-derived NSCs injected into laboratory animals with brain lesions were shown to migrate to the lesions and some motor function improvement was observed.
Cardiomyocytes
Beating cardiac muscle cells, iPSC-derived
cardiomyocytes
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 tha ...
, can be mass-produced using chemically defined differentiation protocols. These protocols typically modulate the same developmental signaling pathways required for
heart development
Heart development, also known as cardiogenesis, refers to the prenatal development of the heart. This begins with the formation of two endocardial tubes which merge to form the tubular heart, also called the primitive heart tube. The heart is t ...
. These iPSC-cardiomyocytes can recapitulate genetic
arrhythmias
Arrhythmias, also known as cardiac arrhythmias, heart arrhythmias, or dysrhythmias, are irregularities in the heartbeat, including when it is too fast or too slow. A resting heart rate that is too fast – above 100 beats per minute in adults ...
and cardiac drug responses, since they exhibit the same genetic background as the patient from which they were derived.
In June 2014, Takara Bio received technology transfer from iHeart Japan, a venture company from Kyoto University's iPS Cell Research Institute, to make it possible to exclusively use technologies and patents that induce differentiation of iPS cells into cardiomyocytes in Asia. The company announced the idea of selling cardiomyocytes to pharmaceutical companies and universities to help develop new drugs for heart disease.
On March 9, 2018, the Specified Regenerative Medicine Committee of Osaka University officially approved the world's first clinical research plan to transplant a "myocardial sheet" made from iPS cells into the heart of patients with severe heart failure. Osaka University announced that it had filed an application with the Ministry of Health, Labor and Welfare on the same day.
On May 16, 2018, the clinical research plan was approved by the Ministry of Health, Labor and Welfare's expert group with a condition.
In October 2019, a group at Okayama University developed a model of ischemic heart disease using cardiomyocytes differentiated from iPS cells.
Red blood cells
Although a pint of donated blood contains about two trillion red blood cells and over 107 million blood donations are collected globally, there is still a critical need for blood for transfusion. In 2014,
type O red blood cell
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 "holl ...
s were synthesized at the Scottish National Blood Transfusion Service from iPSC. The cells were induced to become a
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 ...
and then
blood cell
A blood cell, also called a hematopoietic cell, hemocyte, or hematocyte, is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), ...
s and then red blood cells. The final step was to make them eject their nuclei and mature properly. Type O can be transfused into all patients. Human clinical trials were not expected to begin before 2016.
Clinical trial
The first human
clinical trial
Clinical trials are prospective biomedical or behavioral research studies on human participants designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as novel vaccines, drugs, dietar ...
using
autologous
Autotransplantation is the transplantation of organs, tissues, or even particular proteins from one part of the body to another in the same person ('' auto-'' meaning "self" in Greek).
The autologous tissue (also called autogenous, autogene ...
iPSCs was approved by the
Japan Ministry Health and was to be conducted in 2014 at the
Riken Center for Developmental Biology in
Kobe
Kobe ( , ; officially , ) is the capital city of Hyōgo Prefecture Japan. With a population around 1.5 million, Kobe is Japan's seventh-largest city and the third-largest port city after Tokyo and Yokohama. It is located in Kansai region, whic ...
. However the trial was suspended after Japan's new regenerative medicine laws came into effect in November 2015.
More specifically, an existing set of guidelines was strengthened to have the force of law (previously mere recommendations).
iPSCs derived from
skin cell
Skin is the layer of usually soft, flexible outer tissue covering the body of a vertebrate animal, with three main functions: protection, regulation, and sensation.
Other animal coverings, such as the arthropod exoskeleton, have different de ...
s from six patients with
wet age-related macular degeneration
Macular degeneration, also known as age-related macular degeneration (AMD or ARMD), is a medical condition which may result in blurred or no vision in the center of the visual field. Early on there are often no symptoms. Over time, however, som ...
were reprogrammed to differentiate into
retinal pigment epithelial (RPE) cells. The cell sheet would be transplanted into the affected
retina
The retina (from la, rete "net") is the innermost, light-sensitive layer of tissue of the eye of most vertebrates and some molluscs. The optics of the eye create a focused two-dimensional image of the visual world on the retina, which then ...
where the degenerated RPE tissue was excised. Safety and vision restoration monitoring were to last one to three years.
In March 2017, a team led by
Masayo Takahashi
is a Japanese medical physician, ophthalmologist and stem cell researcher.
Takahashi serves as a project research leader at the Riken Center for Developmental Biology in Kobe focusing on the clinical application of iPS Cell (induced Plurip ...
completed the first successful transplant of iPS-derived retinal cells from a donor into the eye of a person with advanced macular degeneration. However it was reported that they are now having complications. The benefits of using autologous iPSCs are that there is theoretically no risk of
rejection and that it eliminates the need to use embryonic stem cells. However, these iPSCs were derived from another person.
[
New clinical trials involving iPSCs are now ongoing not only in Japan, but also in the US and Europe. Research in 2021 on the trial registry Clinicaltrials.gov identified 129 trial listings mentioning iPSCs, but most were non-interventional.
]
Strategy for obtaining universal iPSCs
To make iPSC-based regenerative medicine technologies available to more patients, it is necessary to create universal iPSCs that can be transplanted independently of haplotype
A haplotype ( haploid genotype) is a group of alleles in an organism that are inherited together from a single parent.
Many organisms contain genetic material ( DNA) which is inherited from two parents. Normally these organisms have their DNA or ...
s of HLA. The current strategy for the creation of universal iPSCs has two main goals: to remove HLA expression and to prevent NK cell
Natural killer cells, also known as NK cells or large granular lymphocytes (LGL), are a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of known innate lymphoid cells (ILC) and represen ...
s attacks due to deletion of HLA. Deletion of the B2M and CIITA
CIITA is a human gene which encodes a protein called the class II, major histocompatibility complex, transactivator. Mutations in this gene are responsible for the bare lymphocyte syndrome in which the immune system is severely compromised and c ...
genes using the CRISPR/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 ...
system has been reported to suppress the expression of HLA class I and class II, respectively. To avoid NK cell attacks. transduction of ligands
In coordination chemistry, a ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's electro ...
inhibiting NK-cells, such as HLA-E
HLA class I histocompatibility antigen, alpha chain E (HLA-E) also known as MHC class I antigen E is a protein that in humans is encoded by the ''HLA-E'' gene. The human HLA-E is a non-classical MHC class I molecule that is characterized by a limit ...
and 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 ...
has been used. HLA-C
HLA-C belongs to the MHC (human = HLA) class I heavy chain receptors. The C receptor is a heterodimer consisting of a HLA-C mature gene product and β2-microglobulin. The mature C chain is anchored in the membrane. MHC Class I molecules, like HL ...
is left unchanged, since the 12 common HLA-C alleles are enough to cover 95% of the world's population.
Anti-aging properties
A multipotent mesenchymal stem cell, when induced into pluripotence, holds great promise to slow or reverse aging phenotypes. Such anti-aging properties were demonstrated in early clinical trials in 2017. In 2020, Stanford University
Stanford University, officially Leland Stanford Junior University, is a private research university in Stanford, California. The campus occupies , among the largest in the United States, and enrolls over 17,000 students. Stanford is consider ...
researchers concluded after studying elderly mice that old human cells when subjected to the Yamanaka factors, might rejuvenate and become nearly indistinguishable from their younger counterparts.
See also
* Induced stem cells
Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotent – ...
* Stem cell treatments
Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition. , the only established therapy using stem cells is hematopoietic stem cell transplantation. This usually takes the form of a bone-marrow transplantation, but ...
* Stimulus-triggered acquisition of pluripotency cell, a now-discredited claim of pluripotent stem cell generation by immersing cells in an acid
* Induced pluripotent stem cells vs embryonic stem cells lines obtained by SCNT (discussion)
* Dedifferentiation
Dedifferentiation (pronounced dē-ˌdi-fə-ˌren-chē-ˈā-shən) is a transient process by which cells become less specialized and return to an earlier cell state within the same lineage. This suggests an increase in a cell potency, meaning that a ...
* Directed differentiation
Directed differentiation is a bioengineering methodology at the interface of stem cell biology, developmental biology and tissue engineering. It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro t ...
* Pluripotency 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 ...
References
External links
Center for iPS Cell Research and Application, Kyoto University
With few factors, adult cells take on character of embryonic stem cells
Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4
2 Minute Video from BSCRF about Induced Pluripotent Stem Cells
20Minute Video / The Discovery and Future of Induced Pluripotent Stem (iPS) Cells by Dr. Yamanaka
January 8, 2008
Fact sheet on reprogramming
University of Oxford practical workshop on pluripotent stem cell technology
* ttps://www.cambioscience.com/ipscjun19-induced-pluripotent-stem-cells-differentiation-to-hepatocytes-3-4-june-2019-cambridge-uk/ CamBioScience iPSC course
{{DEFAULTSORT:Induced Pluripotent Stem Cell
Stem cells
Induced stem cells
2006 in biotechnology