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Induced pluripotent stem cells (iPSCs) are artificial stem cells produced
from somatic cells through co-expression of defined pluripotency-
associated factors. Like embryonic stem cells (ESCs), they can typically
proliferate and self-renew indefinitely in vitro and differentiate into
derivatives of all three primary germ layers (i.e., ectoderm, mesoderm,
and endoderm) as well as germ cells that give rise to the gametes.
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In 1996, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin Institute in
Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first cloned mammal.
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The Dolly experiment showed that scientists could reprogram
the nucleus of somatic cells by transferring the contents of
the nucleus into oocytes that have had their nuclei removed, a
technique called somatic cell nuclear transfer (SCNT).
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In 2006, Takahashi and Yamanaka selected twenty-four candidate genes as factors that they
hypothesized could possibly induce somatic cells to become pluripotent, and they began to test them
one at a time.
The newly inserted gene endowed mice with resistance to an antibiotic named G418. The researchers
labeled the resulting retroviruses mixed with host DNA as retroviral factors.
Takahashi and Yamanaka placed the retrovirus-infected cells into cell culture with G418 antibiotic and
cells to provide nourishment, called feeder cells. If one of the infected cells showed G418 resistance,
then the scientists would know that one of the twenty-four genes influenced the cell to become an
embryonic stem cell-like cell. However, none of the cells showed a resistance to G418, so Takahashi
and Yamanaka reworked their approach.
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Tested 24 candidate genes that are important to maintaining pluripotency and identity in ES
cells
• Used gene targeting to replace Fbx15 gene with βgeo cassette granting G418 (antibiotic)
resistance
• Assumes that activation of Fbx15 locus really does correlate with pluripotency
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Yamanaka et al, 2006
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Next, Takahashi tried to insert into a fibroblast cell multiple retroviral
factors instead of one at a time. The researchers added all of the twenty-
four retroviral factors at the same time into mouse fibroblast cells. This time,
there were twenty-two cell colonies that showed a resistance to G418, meaning
that there were colonies in which the cells exhibited embryonic stem cell
properties.
After examination, Takahashi and Yamanaka concluded that the cells were
similar to embryonic stem cells and duplicated themselves in similar periods of
as embryonic stem cells. They named the cells iPS-MEF24, signifying
pluripotent stem cells induced from mouse embryonic fibroblasts by twenty-
four factors.
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The next experiments aimed to identify specific factors responsible for the generation of
iPS cells. To isolate these specific factors, the researchers removed retroviral factors one
at a time from the original twenty-four, and each time they removed a factor, they
repeated their cell colony procedures.
If the researchers removed a factor and the resultant cell colony wasn't resistant to
antibiotics, they knew that the missing factor somehow influenced the generation of iPS
cells.
Takahashi and Yamanaka found that of the ten genes, when they combined four genes in
particular (Oct3/4, KIf4, Sox2, and c-Myc), they produced the most cells that were like
embryonic stem cells. The scientists named the cells iPS-MEF4. Takahashi and
Yamanaka deemed those four genes important in the role of iPS cell generation. They
concluded that iPSCs are similar, but not identical to embryonic stem cells.
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Kruppel-like factor 4 (KLF4; gut-enriched Krüppel-like factor or GKLF) is a member of
the KLF family of zinc finger transcription factors, which belongs to the relatively large
family of SP1-like transcription factors KLF4 is involved in the regulation
of proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also
suggests that KLF4 is a tumor suppressor in certain cancers, including colorectal cancer.
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, l-myc, and n-myc. c-myc
was the first gene to be discovered in this family, due to homology with the viral gene v-
myc.
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After their mouse experiments, in 2007 Takahashi and Yamanaka published the
results of another experiment that detailed methods and results used to produce
iPS cells with human cells.
The union of both cocktails, yielding six RFs (OCT4, KLF4, SOX2, MYC,
LIN28 and NANOG), is sometimes used to enhance reprogramming efficiency
https://embryo.asu.edu/pages/induced-pluripotent-stem-cell-experiments-kazutoshi-takahashi-and-shinya-yamanaka-
2006-and#:~:text=The%20Dolly%20experiment%20showed%20that,cell%20nuclear%20transfer%20(SCNT).
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In 2012, Yamanaka and John Gurdon received the Nobel Prize in Physiology or Medicine
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Schematic depiction of production of mice using iPS cells and tetraploid complementation.
Retrovirus-mediated transfection with four transcription factors (Oct4, Sox2, Klf4, and c-Myc)
into mouse embryonic fibroblasts (MEF) has resulted in the generation of induced pluripotent
stem (iPS) cells. Then iPS cells are injected into tetraploid blastocysts to generate iPSC mice
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Normal mammalian somatic cells are diploid: each chromosome (and thus every gene) is present
in duplicate (excluding genes from X chromosome absent in Y chromosome).
The assay starts with producing a tetraploid cell in which every chromosome exists fourfold. This
is done by taking an embryo at the two-cell stage and fusing the two cells by applying an
electrical current. The resulting tetraploid cell will continue to divide, and all daughter cells will
also be tetraploid.
Such a tetraploid embryo can develop normally to the blastocyst stage and will implant in the wall
of the uterus. The tetraploid cells can form the extra-embryonic tissue (placenta, etc.); however, a
proper fetus will rarely develop.
In the tetraploid complementation assay, one now combines such a tetraploid embryo (either at
the morula or blastocyst stage) with normal diploid embryonic stem cells (ES). The embryo will
then develop normally; the fetus is exclusively derived from the ES cell, while the extra-
embryonic tissues are exclusively derived from the tetraploid cells.
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The tetraploid complementation assay is also used to test
whether induced pluripotent stem cells (stem cells artificially
produced from differentiated cells, e.g. from skin cells) are as
competent as normal embryonal stem cells.
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Generation and applications of iPSCs. a Various methods and approaches are used to convert somatic
cells into iPSCs. Integrative methods such as integrative viruses and vectors provide the highest
reprogramming efficiency but the lowest safety. In contrast, non-integrative approaches such as the use
of small molecules and microRNAs tend to have a less reprogramming efficiency.
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Limitations:
Viruses and/or integrative methods usually have the highest reprogramming efficiency but the
lowest degree of safety (e.g., insertional mutagenesis and presence of viral components). In
contrast, safer approaches such as the use of small molecules either have less reprogramming
efficiency or usually cannot induce pluripotency alone and are therefore frequently used in
combination(s) with classical reprogramming factors.
Genomic integration may lead to disruption of tumor suppressor genes and/or aberrant permanent
activation of proto-oncogenes, thereby potentially giving rise to the malignant transformation of
the genetically modified cells. This is particularly true when retroviruses are used, since they
tend to randomly integrate into the host cell’s genome.
Alternatively, the pluripotency genes used to generate iPSCs (in particular, the proto-
oncogenes c-MYC ) may later become re-activated in the transplanted cells differentiated from
iPSCs.
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The safety of the reprogramming strategy used for iPSC establishment is not a
significant concern when iPSCs are used for applications other than regenerative
medicine. Therefore, integrative methods, which offer the advantage of high
reprogramming efficiency, could be employed in disease modeling, drug screening,
and basic research.
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Therefore, significant preference lies in using reprogramming (and
differentiation) strategies that exhibit faster kinetics in order to minimize
culture-induced epigenetic and genetic changes.
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Applications
Patient derived neural cells have the specific advantage of retaining the genetic
background of the donor and thus offer a unique in vitro neuronal disease model.
They are an unlimited source of cells that allows the analysis of the cellular
mechanisms involved in disease. Furthermore, they provide a novel platform to test
new drugs and genetic therapies as well as a source of cells that could potentially be
used for cell replacement therapy.
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Use of patient-derived induced pluripotent stem cells (iPSCs) for modeling genetic neurological
diseases.
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Genetic screening of patients affected by a neurological disorder may lead to the
identification of specific mutations causing disease. Patient-derived somatic cells (fibroblast
and other cell types) can be then reprogrammed to a pluripotent state.
iPSCs carrying the disease-related mutation (indicated in red) can be then differentiated into
the neural cells type (neurons and glial cells) which are affected in the disease.
This allows for the in vitro study of the molecular mechanisms downstream the genetic
mutation. In order to overcome genetic background variability and to validate the effect of
the genetic mutation on phenotype observed in vitro, isogenic control iPSCs can be
generated via genomic correction of the mutation.
Moreover, in vitro differentiated cells can be used for high-throughput screening of drugs or
the validation of specific genetic therapies that can then be translated into clinical practice.
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Potential applications of induced pluripotent stem cells for study of Parkinson’s disease. Gene editing technology
enables one to generate knockin mutant Parkinson’s disease (PD) iPSCs and isogenic control cell lines. Dopamine (DA)
neurons can be successfully differentiated from iPSCs. Both patient-derived and gene-editing iPSCs could be powerful
tools for modeling PD for mechanistic studies and drug discovery. Healthy or corrected iPSCs could serve as normal
controls and cell sources for cell therapy. Ke M, Chong CM, Su H. Using induced pluripotent stem cells for modeling
Parkinson’s
1 1 / 2 1 / 2 0 2 disease. World
1 J Stem Cells 2019; 11(9): 634-649 [PMID: 31616540 DOI: 10.4252/wjsc.v11.i9.634] 40
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A Bug’s Life – CRISPR technology was discovered in
bacteria
The term CRISPR (clustered regularly interspaced short palindromic repeats) was coined in
2002 by Dutch scientists working on bacteria genomes. It describes regions of the genome
that contain multiple copies of DNA repeats separated by unique ‘spacers’.
Though its discoverers had few clues at the time about what these curious sequences do, it
has become apparent that these DNA elements represent a form of bacterial immunity against
pathogens like viruses.
Much like our own immune system, CRISPR regions store the memory of previous
infections and help producing a stronger response when invaded again. Viral DNA seized by
the bacteria upon initial infection is incorporated into the CRISPR regions to become the
spacers.
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• These hybrid chunks of native (repeats) and foreign
(spacers) sequences are then expressed in the form of
short RNA molecules. These aptly named ‘guide
RNAs’ can form a larger ‘seek and destroy’ structure
when conjoined with an enzyme called Cas9.
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