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Present and future challenges of induced pluripotent stem cells
Mari Ohnuki, Kazutoshi Takahashi. Published 28 September 2015.DOI: 10.1098/rstb.2014.0367 and https://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/popular-medicineprize2012.pdf
Our body consists of more than 200 committed cell types,
some of which work independently, o eg blood cells,
others form tissues and work in networks,
o eg synapses from the brain to the end of the body
great diversity, but all the cells in our body evolve from a unicellular zygote.
A zygote → morula → blastocyst through mitotic cell division before implantation.
Inner cell mass (ICM) → an epiblast of the post-implantation embryo → three germ layers: the endoderm, mesoderm or ectoderm.
The ICM can differentiate into all the cell types in the human body
this is pluripotency
first cultured as embryonic stem cells (ESCs). The fertilised egg can give rise to all other cell types,
A characteristic called totipotency
(from Latin totus – “whole”, “entire” and potentia – “ability”, “power”).
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Pluripotent cells
(plures – “several”)
can develop into all types of cells except those that form the amniotic sac and the placenta.
An early embryo consists mainly of pluripotent stem cells.
Multipotent cells can develop into any of a family of closely related cell types.
Eg, the blood of adult humans contains multipotent stem cells o can develop into various types of blood cells
o but not into neurons. Embryonic Stem Cells - ESCs
Used to make genetically engineered mice.
Combined with homologous recombination technology o gene deficient mice
knockout mice
Normal embryonic development with cellular differentiation
Had been thought of as one-way - wrong
undifferentiated stem or progenitor cell state to a physiologically mature cell
once believed unneeded genetic code in committed cells other than germ cells was lost o the Weismann barrier - wrong
Clone a frog through the nuclear transfer of embryonic cells
1952
Robert Briggs and Thomas Joseph King
Couldn’t do it in somatic cells
irreversible changes took place in the somatic nuclei - wrong Clone a frog through the nuclear transfer of somatic cells.
1962
John Gurdon
reprogramming of cell fate promptly after the emergence of Waddington's dogma.
a cloned sheep in the late-twentieth century
erasing of epigenetic memories in somatic nuclei is possible, even in mammals
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Nuclear Transplantation
John B. Gurdon
1. destroyed the nucleus in a frog’s egg
2. replaced it with a nucleus taken from a mature cell from a tadpole 3. modified egg cell developed into a normal tadpole
4. laterexperiments involving transferral of cell nuclei have resulted in the cloning of several different mammals
Cell fate changes on Waddington's epigenetic landscape.
Waddington’s original picture. Cell at the top can go down many paths, but it cannot go up.
Using Waddington’s picture to show how undifferentiated cells become differentiated
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Pluripotent stem cells can commit to various somatic cell lines though a middle, progenitor state during embryonic development and in vitro differentiation.
Direct reprogramming, or transdifferentiation, using tissue-specific transcription factors converts the fate of lineage-committed cells (bottom left) to another differentiated fate (bottom centre), bypassing the need for a pluripotent state.
There are several ways of reprogramming lineage-committed cells (bottom right) toward pluripotency (top).
Reprogramming factors
discovered 1987
Davis et al.
complementary DNA subtraction
three genes that were expressed mostly in the proliferative myoblasts
o one of them was myogenic differentiation 1 (MYOD1, also known as MYOD), o encoded a basic-helix–loop–helix transcription factor
o forced expression of Myod1 is enough to convert fibroblasts to myosin-expressing stable myoblasts
Pluripotent cells
a specific set of genes are active
encode proteins called transcription factors
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Shinya Yamanaka
2006
selected 24 of the genes that were associated with pluripotency
inserted them into fibroblast cells o one gene at a time
o with the help of a virus
Nothing happened. Inserted all 24 genes at once
some of the cells converted into something like embryonic stem cells.
repeated the experiment
gradually reducing the number of genes inserted Finally a surprisingly simple recipe
only four of the genes were required to get the fibroblasts to become pluripotent stem cells.
Shinya Yamanaka studied genes that are important for the function of stem cells. When he inserted four of these genes (1) into fibroblast cells from mouse skin (2), the fibroblasts were reprogrammed and became pluripotent stem cells (3), which could develop into all the different cell types in a mouse. He called these cells induced pluripotent stem cells (iPS cells).
4 transcription factors (Myc, Oct3/4, Sox2 and Klf4) were enough to convert mouse embryonic or adult fibroblasts to pluripotent stem cells.
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Many improvements have been made to the technology.
1. The delivery mechanism of pluripotency factors has been improved.
At first retroviral vectors, that integrate randomly in the genome and cause deregulation of genes that contribute to tumor formation, were used.
o Now, non-integrating viruses, o stabilised RNAs or proteins, or
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2. Transcription factors required for inducing pluripotency in different cell types have been identified (e.g. neural stem cells).
3. Small substitutive molecules were identified, that can substitute for the function of the transcription factors.
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4. Transdifferentiation experiments were carried out. They tried to change the cell fate without proceeding through a pluripotent state.
systematically identified genes that carry out transdifferentiation using combinations of transcription factors that induce cell fate switches.
found trandifferentiation within germ layer and between germ layers, e.g., o exocrine cells to endocrine cells,
o fibroblast cells to myoblast cells, o fibroblast cells to cardiomyocyte cells, o fibroblast cells to neurons
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(a) Cell transplantation therapy
May help treatment of intractable diseases such as Parkinson's disease, spinal cord injuries, and dry age-related macular degeneration (AMD).
In 2007, iPSCs was reported to treat a mouse model of sickle-cell anaemia, (b) Disease modelling and drug screening
Limitations of animal models in showing human diseases.
Eg. drugs for amyotrophil lateral sclerosis work in mice and rats, but are ineffective in the treatment of human patients.
o Amyotrophic lateral sclerosis (ALS), o Rett syndrome,
o spinal muscular atrophy (SMA), o α1-antitrypsin deficiency,
o familial hypercholesterolemia and glycogen storage disease type 1A. - For cardiovascular disease,
o Timothy syndrome, o LEOPARD syndrome,
o type 1 and 2 long QT syndrome - Alzheimer’s, o Spinocerebellar ataxia,
o Huntington’s etc.
(c) iPS cells provide screening platforms for development and validation of therapeutic compounds.
kinetin was a novel compound found in iPS cells from familial dysautonomia
beta blockers & ion channel blockers for long QT syndrome were identified with iPS cells.
In 2013, iPS cells were used to generate a human vascularized and functional liver in mice in Japan. Multiple stem cells were used to differentiate the component parts of the liver, which then self-organized into the complex structure. When placed into a mouse host, the liver vessels connected to the hosts vessels and performed normal liver functions, including breaking down of drugs and liver secretions.
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Advantages of iPSC technology
The ethical issues are eliminated by the use of iPSCs
Reduced chances of immunorejection
Throughput screening for predicting toxicity/therapeutic responses of newly developed drugs
o prediction of toxicity and possible side effects of newly developed drugs in different body cells
Lowering the overall cost and risk of clinical trials
Development of a personalized approach for administration of drugs
Gene targeting and correction technologies (gene therapy)
For disease modeling, the phenotypes need to be consistent every time, which is a possibility in case of iPSCs
