Induced pluripotent stem cells, commonly abbreviated as
iPS cells or
iPSCs are a type of pluripotent
stem cellThis article is about the cell type. For the medical therapy, see Stem Cell TreatmentsStem cells are biological cells found in all multicellular organisms, that can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells...
artificially derived from a non-pluripotent cell, typically an adult
somatic cellA somatic cell is any biological cell forming the body of an organism; that is, in a multicellular organism, any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell...
, by inducing a "forced" expression of specific
geneA gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s.
Induced Pluripotent Stem Cells are similar to natural pluripotent stem cells, such as
embryonic stem (ES) cellsEmbryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
, in many respects, such as the expression of certain stem cell genes and proteins,
chromatin methylationDNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
patterns, doubling time,
embryoid bodyEmbryoid bodies are aggregates of cells derived from embryonic stem cells, and have been studied for years with mouse embryonic stem cells. Cell aggregation is imposed by hanging drop, plating upon non-tissue culture treated plates or spinner flasks; either method prevents cells from adhering to a...
formation,
teratomaA teratoma is an encapsulated tumor with tissue or organ components resembling normal derivatives of all three germ layers. There are rare occasions when not all three germ layers are identifiable...
formation, viable
chimeraA chimera or chimaera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic...
formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed.
iPSCs were first produced in 2006 from mouse cells and in 2007 from human cells. This has been cited as an important advance in stem cell research, as it may allow researchers to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the
controversialThe stem cell controversy is the ethical debate primarily concerning the creation, treatment, and destruction of human embryos incident to research involving embryonic stem cells. Not all stem cell research involves the creation, use, or destruction of human embryos...
use of embryos. Because iPSCs are developed from a patient's own somatic cells, it was believed that treatment of iPSCs would avoid any immunogenic responses; however, Zhao et al. have challenged this assumption.
Depending on the methods used, reprogramming of adult cells to obtain iPSCs may pose significant risks that could limit its use in humans. For example, if viruses are used to genomically alter the cells, the expression of cancer-causing genes or oncogenes may potentially be triggered. In February 2008, in ground-breaking findings published in the journal
CellCell is a peer-reviewed scientific journal publishing research papers across a broad range of disciplines within the life sciences. Areas covered include molecular biology, cell biology, systems biology, stem cells, developmental biology, genetics and genomics, proteomics, cancer research,...
, 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 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 anchorsCell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargo . The "cargo" is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions...
was sufficient to induce pluripotency. The acronym given for those iPSCs is
piPSCs (protein-induced pluripotent stem cells).
Production of IPSCs
iPS cells are typically derived by
transfectionTransfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
of certain stem cell-associated genes into non-pluripotent cells (although this technique is becoming less popular since it is known to be prone to cancer formation), such as adult
fibroblastA fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for animal tissues, and plays a critical role in wound healing...
s. Transfection is typically achieved through viral vectors, such as
retrovirusA retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
es. Transfected genes include the master transcriptional regulators Oct-3/4 (Pou5f1) and
Sox2SRY -box 2, also known as SOX2, is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells....
, although it is suggested that other genes enhance the efficiency of induction. After 3–4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a
reporter geneIn molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
and antibiotic selection.
First generation
Induced pluripotent stem cells were first generated by
Shinya Yamanakais a Japanese physician and adult stem cell researcher. He serves as the director of Center for iPS Cell Research and Application and a professor at the Institute for Frontier Medical Sciences at Kyoto University, as a senior investigator at the UCSF-affiliated J...
's team at
Kyoto University, or is a national university located in Kyoto, Japan. It is the second oldest Japanese university, and formerly one of Japan's Imperial Universities.- History :...
, Japan in 2006. Yamanaka used genes that had been identified as particularly important in embryonic stem cells (ESCs), and used
retrovirusA retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
es to transduce mouse fibroblasts with a selection of those genes. Eventually, four key pluripotency genes essential for the production of pluripotent stem cells were isolated; Oct-3/4, SOX2, c-
MycMyc is a regulator gene that codes for a transcription factor. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences and recruiting histone acetyltransferases...
, and
Klf4Krueppel-like factor 4 is a protein that in humans is encoded by the KLF4 gene.. In embryonic stem cells , KLF4 has been demonstrated to be a good indicator of stem-like capacity. It is suggested that the same is true in mesenchymal stem cells ....
. Cells were isolated by antibiotic selection of
Fbx15Fbx15, otherwise known as Fbxo15, is a protein expressed in undifferentiated embryonic stem cells.It is expressed during coexpression of Oct3/4 and SOX2, two genes identified to be important in embryonic stem cell self-renewal and differentiation repression...
+ cells. However, this iPS cell line showed DNA methylation errors compared to original patterns in ESC lines and failed to produce viable
chimeraA chimera or chimaera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic...
s if injected into developing embryos.
Second generation in mice
In June 2007, the same group published a breakthrough study along with two other independent research groups from Harvard, MIT, and the
University of California, Los AngelesThe University of California, Los Angeles is a public research university located in the Westwood neighborhood of Los Angeles, California, USA. It was founded in 1919 as the "Southern Branch" of the University of California and is the second oldest of the ten campuses...
, showing successful reprogramming of mouse fibroblasts into iPS cells and even producing viable chimera. These cell lines were also derived from mouse fibroblasts by retroviral mediated reactivation of the same four endogenous pluripotent factors, but the researchers now selected a different marker for detection. Instead of Fbx15, they used
NanogThe North American Network Operators' Group is an educational and operational forum for the coordination and dissemination of technical information related to backbone/enterprise networking technologies and operational practices. It runs meetings, talks, surveys, and an influential mailing list...
which is an important gene in ESCs. DNA methylation patterns and production of viable chimeras (and thereby contributing to subsequent germ-line production) indicated that Nanog is a major determinant of cellular pluripotency.
Unfortunately, one of the four genes used (namely, c-Myc) is oncogenic, and 20% of the chimeric mice developed cancer. In a later study, Yamanaka reported that one can create iPSCs even without c-Myc. The process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer.
Two-fathered mice
Reproductive scientists in University of Texas MD Anderson Cancer Center have created mice with
nuclear DNANuclear DNA, nuclear deoxyribonucleic acid , is DNA contained within a nucleus of eukaryotic organisms. In mammals and vertebrates, nuclear DNA encodes more of the genome than the mitochondrial DNA and is composed of information inherited from two parents, one male, and one female, rather than...
(nDNA) solely from two fathers, using iPS technology. Foetal
fibroblastA fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for animal tissues, and plays a critical role in wound healing...
s from one father (XY) were cultivated and one percent of the resultant cells had spontaneously lost a Y-chromosome; like an individual with
Turner SyndromeTurner syndrome or Ullrich-Turner syndrome encompasses several conditions in human females, of which monosomy X is most common. It is a chromosomal abnormality in which all or part of one of the sex chromosomes is absent...
(X0). These cells were injected in female
blastocystThe blastocyst is a structure formed in the early embryogenesis of mammals, after the formation of the morula. It is a specifically mammalian example of a blastula. It possesses an inner cell mass , or embryoblast, which subsequently forms the embryo, and an outer layer of cells, or trophoblast,...
s (XX), which gestated in surrogate mothers to form female
chimeraA chimera or chimaera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic...
s (X0/XX). When these mated with male mice (XY). Some of the offspring had nDNA from the original father and also from the mated male but not from the female blastocysts or the surrogate mother. Both male and female two-fathered mice were viable.
Human induced pluripotent stem cells
In November 2007, a milestone was achieved by creating iPSCs from adult human cells; two independent research teams' studies were released - one in
ScienceScience is the academic journal of the American Association for the Advancement of Science and is one of the world's top scientific journals....
by
James ThomsonJames Alexander Thomson is an American developmental biologist best known for deriving the first human embryonic stem cell line in 1998 and for deriving human induced pluripotent stem cells in 2007.-Thomson's research:...
at
University of Wisconsin–MadisonThe University of Wisconsin–Madison is a public research university located in Madison, Wisconsin, United States. Founded in 1848, UW–Madison is the flagship campus of the University of Wisconsin System. It became a land-grant institution in 1866...
and another in
CellCell is a peer-reviewed scientific journal publishing research papers across a broad range of disciplines within the life sciences. Areas covered include molecular biology, cell biology, systems biology, stem cells, developmental biology, genetics and genomics, proteomics, cancer research,...
by Shinya Yamanaka and colleagues at Kyoto University, Japan. With the same principle used earlier in mouse models, Yamanaka had successfully transformed human fibroblasts into pluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a
retroviralA retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
system. Thomson and colleagues used OCT4, SOX2, NANOG, and a different gene LIN28 using a
lentiviralLentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period...
system.
Limitations of the transcription factor approach
Although the traditional method using transcription factors such as Oct3/4,
Sox2SRY -box 2, also known as SOX2, is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells....
, c-Myc, etc. pioneered by Yamanaka and Thompson was good proof of concept that
somaticThe term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
cells can be reprogrammed to
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells, there are still many key challenges for this method to overcome:
- Throughput: the throughput of successfully reprogrammed cells has been incredibly low. For example, the rate at which somatic
The term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
cells were reprogrammed into iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells in the Yamanaka mouse study was .01-.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 and/or epigenetic changes in the original somaticThe term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
cell population or in the prolonged culture.
- Genomic Insertion: genomic integration of the transcription factors limits the utility of the transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
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. plasmids , adenovirus es, and transposonTransposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
vectors have all been explored, but these often come with the tradeoff of lower throughput.
- Tumors: another main challenge was mentioned above—some of the reprogramming factors are oncogenes that bring on a potential tumor risk. Inactivation or deletion of the tumor suppressor p53, which is the master 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 that synthesizes the extracellular matrix and collagen, the structural framework for animal tissues, and plays a critical role in wound healing...
(MEF)-derived iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells that could be injected into tetraploid blastocysts and resulted in the live birth of mice derived entirely from iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells, thus ending the debate over the equivalence of embryonic stem cells (ESCs) and iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
with regard to pluripotency. However, some of the other techniques profiled below demonstrated limited or incomplete equivalent to ESCs.
The table at right summarizes the key strategies and techniques used to develop
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells over the past half-decade. Rows of similar colors represents studies that used similar strategies for reprogramming.
Mimicking transcription factors with small compounds
One of the main strategies for avoiding problems (1) and (2) has been to use small compounds that can mimic the effects of transcription factors. These molecule 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 factorIn molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
method). The researchers proposed that this compound was mimicking the signaling that is usually caused by the
transcription factorIn molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
c-Myc.
A similar type of compensation mechanism was proposed to mimic the effects of
Sox2SRY -box 2, also known as SOX2, is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells....
. In 2008, Ding et al. used the inhibition of histone methyl transferase (HMT) with BIX-01294 in combination with the activation of calcium channels in the plasma membrane in order to increase reprogramming efficiency.
It is foreseeable that such experiments will continue to find small compounds that improve efficiency rates. Ultimately, the goal is to discover a cocktail of reprogramming factors and compounds that efficiently and reliably reprogram
somaticThe term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
cells to
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells.
Alternate vectors
Another key strategy for avoiding problems such as tumor genesis and low throughput has been to use alternate forms of vectors: adenovirus, plasmids, and naked DNA and/or protein compounds.
In 2008, Hochedlinger et al. used an adenovirus 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 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. More recently (in 2009), Freed et al. demonstrated successful reprogramming of human fibroblasts to
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells
. Another advantage of using adenoviruses 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. once again made a huge contribution to the field of
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells with the finding 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, Klf4, and
Sox2SRY -box 2, also known as SOX2, is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells....
).
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 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
transposonTransposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
system. The lifecycle of this system is shown below. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving any footprint mutations in the host cell genome. As you can see in the figure, the piggyBac
transposonTransposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
system involves the re-excision of exogenous genes, and this is what prevents this method from causing issues like insertional mutagenesis
Drug-Like chemicals, recombinant proteins
In 2009, Ding and colleagues demonstrated a successful alternative to
transcription factorIn molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
reprogramming through the use of drug-like chemicals. This was the first method in human cells that was mechanism-specific for the reprogramming process. Ding tackled the problem of genomic insertion by using purified proteins to transform adult cells into embryonic-like cells. Once his team successfully demonstrated this, they tackled the efficiency problem.
Ding’s overall strategy involved biomimicry. He studied the naturally occurring process of
MET-In the arts:* Metropolitan Opera in Manhattan, New York* Metropolitan Museum of Art in Manhattan, New York* Various buildings known as the Metropolitan Opera House* The Metropolitan Ensemble Theater in Kansas City, Missouri-In computing and the Internet:...
(mesenchymal to epithelial cell transition), in which fibroblasts are pushed to a stem-cell like state. Ding first looked for drug-like molecules that inhibited compounds known to be involved in the MET process; these compounds included TGFb (transforming growth factor beta) and MEK (mitogen-activated protein kinase). Ding identified the most active molecules and then studied their effects on
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
creation when used singly or in combination. He concluded that there are two chemicals—ALK5 inhibitor SB431412 and MEK inhibitor PD0325901, which when used in combination are highly effective at promoting the transformation from fibroblast to
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cell.
Although this two-chemical technique bested the efficiency of the classical genetic method by 100fold, Ding sought to do better. He continued with the use of the biomimicry strategy, enlisting another natural pathway—the cell survival pathway. After screening several compounds that target this pathway, the team focused on a new compound called Thiazovivin. Using this protein with the two previous chemicals, the team beat the efficiency of the classic method by 200 fold. Furthermore, this method took only two weeks to complete reprogramming while the classic method took four weeks. Another advantage of Ding’s method was that it overcame the genetic insertion problem because the drug-like molecules were based on natural biological processes.
RNA molecules
Studies by Blelloch et al. in 2009 demonstrated that expression of ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhances the efficiency of induced pluripotency by acting downstream of c-Myc
.
More recently (in April 2011), Morrisey et al. demonstrated another method using microRNA that improved the efficiency of reprogramming to a rate similar to that demonstrated by Ding. MicroRNAs are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Morrisey’s team worked on microRNAs in lung development, and hypothesized that their microRNAs perhaps blocked expression of repressors of Yamanaka’s four transcription factors.
Genes of induction
The generation of iPS cells is crucially dependent on the genes used for the induction.
Oct-3/4 and certain members of the
Sox gene familySOX 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. This HMG box is a DNA binding domain that is highly conserved throughout eukaryotic species...
(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 familyThe Krüppel-like family of transcription factors , so named for their homology to the Drosophila melanogaster Krüppel protein, have been extensively studied for their roles in cell proliferation, differentiation and survival, especially in the context of cancer . All KLF family members are...
(Klf1, Klf2, Klf4, and Klf5), the
Myc familyMyc is a regulator gene that codes for a transcription factor. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences and recruiting histone acetyltransferases...
(c-myc, L-myc, and N-myc),
NanogThe North American Network Operators' Group is an educational and operational forum for the coordination and dissemination of technical information related to backbone/enterprise networking technologies and operational practices. It runs meetings, talks, surveys, and an influential mailing list...
, and
LIN28Lin-28 homolog A is a protein that in humans is encoded by the LIN28 gene. It is a marker of undifferentiated human embryonic stem cells and has been used to enhance the efficiency of the formation of induced pluripotent stem cells from human fibroblasts...
, have been identified to increase the induction efficiency.
- Oct-3/4 (Pou5f1): Oct-3/4 is one of the family of octamer ("Oct") transcription factor
An octamer transcription factor is a transcription factor which binds to the "ATTTGCAT" sequence.Examples include:* Oct-1 - * Oct-2 - * Oct-3/4 – * Oct-6 – * Oct-7 – * Oct-8 - * Oct-9 – * Oct-11 –...
s, and plays a crucial role in maintaining pluripotency. The absence of Oct-3/4 in Oct-3/4+ cells, such as blastomereA blastomere is a type of cell produced by division of the egg after fertilization.- References :* "Blastomere." Stedman's Medical Dictionary, 27th ed. . ISBN 0-683-40007-X...
s and embryonic stem cellEmbryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
s, leads to spontaneous trophoblastTrophoblasts are cells forming the outer layer of a blastocyst, which provide nutrients to the embryo and develop into a large part of the placenta. They are formed during the first stage of pregnancy and are the first cells to differentiate from the fertilized egg...
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, fail to elicit induction, thus demonstrating the exclusiveness of Oct-3/4 to the induction process.
- Sox family
SOX 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. This HMG box is a DNA binding domain that is highly conserved throughout eukaryotic species...
: The Sox family of genes 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 Sox2SRY -box 2, also known as SOX2, is a transcription factor that is essential to maintain self-renewal of undifferentiated embryonic stem cells....
was the initial gene used for induction by Yamanaka et al., Jaenisch et al., and Thomson et al., other genes in the Sox family have been found to work as well in the induction process. Sox1SOX1 is a transcription factor in the Sox protein family. SOX1 expression is restricted to neuroectoderm in the tetrapod embryo. SOX1 is involved in early central nervous system development, where it is functionally redundant with SOX3 and to a lesser degree SOX2, and maintenance of neural...
yields iPS cells with a similar efficiency as Sox2, and genes Sox3Transcription factor SOX-3 is a protein that in humans is encoded by the SOX3 gene.This gene encodes a member of the SOX family of transcription factors involved in the regulation of embryonic brain development and in determination of cell fate. The encoded protein acts as a transcriptional...
, Sox15Protein SOX-15 is a protein that in humans is encoded by the SOX15 gene.-Further reading:...
, and Sox18Transcription factor SOX-18 is a protein that in humans is encoded by the SOX18 gene....
also generate iPS cells, although with decreased efficiency.
- Klf family
The Krüppel-like family of transcription factors , so named for their homology to the Drosophila melanogaster Krüppel protein, have been extensively studied for their roles in cell proliferation, differentiation and survival, especially in the context of cancer . All KLF family members are...
: Klf4Krueppel-like factor 4 is a protein that in humans is encoded by the KLF4 gene.. In embryonic stem cells , KLF4 has been demonstrated to be a good indicator of stem-like capacity. It is suggested that the same is true in mesenchymal stem cells ....
of the Klf family of genes 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. Klf2Krueppel-like factor 2 is a protein that in humans is encoded by the KLF2 gene....
and Klf4 were found to be factors capable of generating iPS cells, and related genes Klf1 and Klf5Krueppel-like factor 5 is a protein that in humans is encoded by the KLF5 gene.-Interactions:KLF5 has been shown to interact with Protein SET....
did as well, although with reduced efficiency.
- Myc family
Myc is a regulator gene that codes for a transcription factor. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences and recruiting histone acetyltransferases...
: The Myc family of genes are proto-oncogenes 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., and unpublished work by Johns Hopkins UniversityThe Johns Hopkins University, commonly referred to as Johns Hopkins, JHU, or simply Hopkins, is a private research university based in Baltimore, Maryland, United States...
reported that c-myc was unnecessary for generation of human iPS cells. 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 teratomaA teratoma is an encapsulated tumor with tissue or organ components resembling normal derivatives of all three germ layers. There are rare occasions when not all three germ layers are identifiable...
s. N-mycV-myc myelocytomatosis viral related oncogene, neuroblastoma derived , also known as MYCN, is a protein which in humans is encoded by the MYCN gene.- Function :...
and L-myc have been identified to induce instead of c-myc with similar efficiency.
- Nanog
The North American Network Operators' Group is an educational and operational forum for the coordination and dissemination of technical information related to backbone/enterprise networking technologies and operational practices. It runs meetings, talks, surveys, and an influential mailing list...
: 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. It is a marker of undifferentiated human embryonic stem cells and has been used to enhance the efficiency of the formation of induced pluripotent stem cells from human fibroblasts...
: LIN28 is an mRNA binding proteinRNA-binding proteins are proteins that bind to RNA. They bind to either double-strand or single-strand RNAs through RNA recognition motif . RNA-binding proteins may regulate the translation of RNA, and post-transcriptional events, such as RNA splicing, editing.They are cytoplasmic and nuclear...
expressed in embryonic stem cellEmbryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
s and embryonic carcinoma cells associated with differentiation and proliferation. Thomson et al. demonstrated it is a factor in iPSC generation, although it is unnecessary.
An open future
The task of producing
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells continues to be challenging due to the five 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. Of course there are a vast number of techniques and methods that have been attempted. Another large set of strategies is to perform a proteomic characterization of
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells. The Wu group at
Stanford UniversityThe Leland Stanford Junior University, commonly referred to as Stanford University or Stanford, is a private research university on an campus located near Palo Alto, California. It is situated in the northwestern Santa Clara Valley on the San Francisco Peninsula, approximately northwest of San...
has made significant headway with this strategy.
Further studies and new strategies should help us find optimal solutions to the five main challenges. An interesting experiment might attempt to combine the good of these strategies into an ultimately effective technique for reprogramming cells to
iPS- Medicine and biology :* Induced pluripotent stem cell* Intermittent photic stimulation, a neuroimaging technique* Intraparietal sulcus, a region of the brain* Ips , a genus of bark beetle- Media and entertainment :...
cells.
Identity
The generated iPSCs were remarkably similar to naturally-isolated pluripotent stem cells (such as mouse and human
embryonic stem cellEmbryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
s, 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 is a non-membrane bound structure composed of proteins and nucleic acids found within the nucleus. Ribosomal RNA is transcribed and assembled within the nucleolus...
and scant cytoplasmThe cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
. 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
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
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 is an enzyme that adds DNA sequence repeats to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. This region of repeated nucleotide called telomeres contains non-coding DNA material and prevents constant loss of important DNA from...
s are necessary to sustain cell division unrestricted by the Hayflick limitThe Hayflick limit is the number of times a normal cell population will divide before it stops, presumably because the telomeres reach a critical length....
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 transcriptaseTelomerase reverse transcriptase is a catalytic subunit of the enzyme telomerase. Its absence is associated with the disorder Cri du chat....
), 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 is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
s, expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence of catecholamineCatecholamines are molecules that have a catechol nucleus consisting of benzene with two hydroxyl side groups and a side-chain amine. They include dopamine, as well as the "fight-or-flight" hormones adrenaline and noradrenaline released by the adrenal medulla of the adrenal glands in response to...
-associated enzymes may indicate that iPSCs, like hESCs, may be differentiable into dopaminergicDopaminergic means related to the neurotransmitter dopamine. For example, certain proteins such as the dopamine transporter , vesicular monoamine transporter 2 , and dopamine receptors can be classified as dopaminergic, and neurons which synthesize or contain dopamine and synapses with dopamine...
neurons. Stem cell-associated genes were downregulated after differentiation.
- Cardiac differentiation: iPSCs were differentiated into cardiomyocytes
Cardiac muscle is a type of involuntary striated muscle found in the walls and histologic foundation of the heart, specifically the myocardium. Cardiac muscle is one of three major types of muscle, the others being skeletal and smooth muscle...
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 is a state in which the immune system's ability to fight infectious disease is compromised or entirely absent. Immunodeficiency may also decrease cancer immunosurveillance. Most cases of immunodeficiency are acquired but some people are born with defects in their immune system,...
mice spontaneously formed teratomaA teratoma is an encapsulated tumor with tissue or organ components resembling normal derivatives of all three germ layers. There are rare occasions when not all three germ layers are identifiable...
s after nine weeks. Teratomas are tumors of multiple lineages containing tissue derived from the three germ layers endodermEndoderm is one of the three primary germ cell layers in the very early embryo. The other two layers are the ectoderm and mesoderm , with the endoderm as the intermost layer...
, mesodermIn all bilaterian animals, the mesoderm is one of the three primary germ cell layers in the very early embryo. The other two layers are the ectoderm and endoderm , with the mesoderm as the middle layer between them.The mesoderm forms mesenchyme , mesothelium, non-epithelial blood corpuscles and...
and ectodermThe "ectoderm" is one of the three primary germ cell layers in the very early embryo. The other two layers are the mesoderm and endoderm , with the ectoderm as the most exterior layer...
; 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
Embryoid bodies are aggregates of cells derived from embryonic stem cells, and have been studied for years with mouse embryonic stem cells. Cell aggregation is imposed by hanging drop, plating upon non-tissue culture treated plates or spinner flasks; either method prevents cells from adhering to a...
”, 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) of blastocyst
The blastocyst is a structure formed in the early embryogenesis of mammals, after the formation of the morula. It is a specifically mammalian example of a blastula. It possesses an inner cell mass , or embryoblast, which subsequently forms the embryo, and an outer layer of cells, or trophoblast,...
s, and in the embryoblast, differentiate into the embryo while the blastocyst’s shell (trophoblastTrophoblasts are cells forming the outer layer of a blastocyst, which provide nutrients to the embryo and develop into a large part of the placenta. They are formed during the first stage of pregnancy and are the first cells to differentiate from the fertilized egg...
) 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 micropipetteA micropipette is one of two different instruments used in science laboratories.-History:The first micropipette was patented in 1960 by Dr Hanns Schmitz . The founder of the company Eppendorf, Dr...
into a trophoblast, and the blastocyst was transferred to recipient females. ChimericA chimera or chimaera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic...
living mouse pups were created: mice with iPSC derivatives incorporated all across their bodies with 10%-90% chimerism.
- Tetraploid complementation
The tetraploid complementation assay is a technique in biology in which cells of two mammalian embryos are combined to form a new embryo. It is used to construct genetically modified organisms, to study the consequences of certain mutations on embryonal development, and in the study of pluripotent...
: 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
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
by preventing the activity of expression proteins or 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 pattern of which cytosins are methylated
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
, 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 megabases 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 alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
s are compacting proteins that are structurally localized to DNA sequences that can effect 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 for Regenerative Medicine
- 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 when injected into immunodeficient mice. Teratoma formation is considered a major obstacle to stem-cell based regenerative medicine by the FDA.
- 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 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 recent paper came out 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.
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