Stem Cells Flashcards
1
Q
What are stem cells?
A
Cells that can differentiate into many different cell types
2
Q
What are the 3 classes of stem cells?
A
- Totipotent
- Pluripotent
- Multipotent
3
Q
Totipotent Stem Cells
A
- Can develop into any cell type in the body, including extra-embryonic tissue (Like placenta)
4
Q
Embryonic Stem cells
A
- Pluripotent
- Derived from the inner cell mass of a blastula
- Can give rise to any cell type in the body, but not to extra-embryonic tissue
- Can be sucked out and expanded in culture (petri dish)
- Given appropriate growth factors, can be made to differentiate into different cell types
- Problem:
o These cells can be taken from/derived from left-over fertilized blastocysts from in vitro fertilization
o BUT they aren’t matched to the patient – so will be a serious amount of rejection from host
5
Q
Multipotent Stem Cells
A
- Founder cells of the 3 germ layers
- Cannot differentiate into cell types from a different germ layer
o i.e. ectoderm stem cell can only differentiate into an ectoderm cell
o e.g. Mesoderm stem cells can give rise to skeletal muscle cells, red blood cells, tubule cells of kidney, but NOT to neurons of brain, or lung cells etc. - Mesenchymal stem cells = stem cells from placenta cord tissue and cord blood
- Hematopoietic stem cells from cord blood: multipotent not pluripotent
6
Q
Discoveries leading to the overturning of the classical view of cell differentiation and developement
A
- John Gurdon reprogrammed adult Xenopus cells in 1950s:
o Took nucleus from adult somatic cell
o Injected into enucleated egg (nucleus removed from egg)
o Allowed development to occur
o Found that it could develop a blastocyst with an inner cell mass
o This is reproductive cloning if it is put back into mother and allowed to develop
o This is a very useful source for generating embryonic stem cells (Therapeutic cloning) - Induced pluripotent stem cells (iPS) by Shinya Yamanaka in 2006
7
Q
Method of SCNT
A
- First shown in 2013
- Took human oocyte with meiotic spindle
- Removed spindle and nucleus and added caffeine (Key step that they discovered)
- Added nucleus of somatic cell into enucleated egg (without spindle)
- Activated oocytes and successfully reprogramed somatic nucleus to form blastocyst
- Has low success rate, so requires large amounts of human oocytes
8
Q
iPS cells
A
- Direct reprogramming of somatic cell into a pluripotent cell by introduction of 4 transcription factors leading to the overexpression of 4 genes (c-myc, oct4, KLF4 and sox2)
- Used fibroblasts instead of oocytes (differentiated somatic cell)
- Overexpression of these 4 genes was sufficient to reprogram cell into an induced pluripotent stem cell (not embryonic stem cells because not from blastocyst)
o Pluripotent because they could be maintained in culture
o Given the right growth factors they coul differentiate into a wide range of cell type from the different germ layers
- Myc: o Proto-oncogene, transcription factor - Oct4: o Proto-oncogene, transcription factor - KLF4: o Kruppel-like factor 4, transcription factor interacts with p53 - Sox2: o SRY box 2, transcription factor, stem cell maintenance in nervous system
9
Q
Ethical issues related to use of human embryos
A
- In order to isolate hESCs from the inner cell mass of a blastocyst, an embryo has to be destroyed and discarded.
o This becomes an ethical issue if you classify life as beginning at conception.
o However, it is only the catholic religion and some protestant denominations that view life as beginning at conception.
o South African legislation allows the use of naturally conceived blastocysts for therapeutic purposes.
o This ethical concern has to be weighed against the potential benefit of utilising surplus IVF embryos that just sit in a freezer. - Safety of the patient.
o Because embryonic stem cells are pluripotent, they have the ability to differentiate into all 3 germ layers.
o If a hESC is transplanted into a patient before it has fully differentiated, risk of developing a teratoma (a tumour with all 3 germ layers present).
o hESCs are cultured in 10% foetal calf serum (the culture also contains other growth factors), and they often remain suspended in this culture for a long time.
o This introduces the risk of those cells being infected with viruses, bacteria or fungi from the bovine serum which can then be transferred to the patient. - Distribution of rights related to drug discovery.
o There is a question mark around who has financial rights if research done on a donated blastocyst leads to the discovery of a drug, do the parents of the donated blastocyst retain any rights to the financial rewards?
10
Q
Ethical issues related to SCNT technology
A
- SCNT cells make use of human oocytes and result in the formation of a blastocyst, which leaves the same ethical concerns related to the destruction of an embryo as there are in hESCs.
o It does however raise new questions about whether a cloned blastocyst has the same moral status as a natural human embryo.
o If your view of when life begins is not that of a catholic or certain protestants, then (like in hESCs) the destruction of the blastocyst will pose no issue.
o South African legislation currently does not allow the use of blastocysts generated by SCNT in research. - Safety of the patient.
o Like hESCs, SCNT cells are pluripotent and have the potential to cause teratomas.
o They are also maintained in culture (although often for less time), and so run the risk of contracting an infection from the bovine serum. - Sourcing of human oocytes.
o Because SCNT has such a low efficiency (±280 oocytes required to generate 1 single patient-specific ntESC) it has the potential to create such a high demand for human oocytes that a commercial market for ova could form.
o The commercial marketing of human material has many ethical questions related to it, and it also has the potential to cause immense harm to the women who are used in this market.
11
Q
Ethical issues related to iPS technology
A
- Because patient-specific iPS cells can be cultured from fibroblasts, there is no need for human embryos. This overcomes the concerns related to the destruction of embryos that is faced by both hESCs and ntESCs -
- Safety of the patient.
o The methylation patterns in iPS cells are less similar to hESCs than those generated by SCNT which leads to questions related to how similar iPS cells really are to hESCs, and whether they are fully pluripotent?
o iPS cells have a much greater genomic instability than both hESCs and ntESCs (due to the oncogenic potential of the 4 proto-oncogenes that are overexpressed), the use of iPS cells can still lead to the formation of teratomas.
o As well as other forms of cancer. - Potential to possibly form human consciousness in culture.
o iPS cells have been shown to be able to form brain organoids that have synchronized neural activity, and the question must be asked if this technology could be used to generate human consciousness.
12
Q
Mitochondrial Replacement Theory
A
- If mother has debilitating mitochondrial disease, she will pass it on to her children, (mitochondrion are maternally inherited)
- MRT can replace faulty mitochondria
- Oocyte from healthy donor female is denucleated
- Metaphase arrested nuclear genome from female with mutant mtDNA is transferred into healthy donor oocyte (cytoplasm only, no nucleus)
- In vitro fertilisation by fathers sperm and develop towards blastocysts which can be implanted back into mother.
- Forms child with 3 parents: father, mother who donated nuclear genome, egg donor who donated healthy mitochondria.
13
Q
Therapeutic Potential of iPS cells
A
- Can be used to directly treat disease, or model disease
- Bypasses many ethical issues around use of human oocytes
- Take skin cells from patient with disease
- Infect with 4 transcription factors
o Generates patient specific iPS cells - Use them to either treat the disease directly or model the disease
- Treat directly:
o Use CRISPR to correct mutations in genes that are shown to be infected
o Repaired iPS cells can differentiate back into healthy cells
o Then transplanted back into patient - Model the disease:
o In vitro differentiation to generate a set of faulty cells
o Use different disease specific drugs to test if you can restore function in these cells
o Once you’ve found the right drugs that work, you can then treat the patient with this drug.
o Can also be used to generate organoids
14
Q
Gene editing with iPS cells
A
- Would get skin cells, gen iPS cells, use CRISPR to correct mutation, get cells to differentiate into blood cells, return healthy blood cells back into patients
Not being used in humans because:
- The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.
- Clinical trials have starter to use iPS cells to treat Age-related Macular Degeneration (AMD)
- Take blood cells, transform into iPS cell
- Differentiate into retinal pigment cells & transplant them into retina of patients and see if you can restore vision
15
Q
Human organoids
A
- Can take iPS cells and get them to deliver into liver, kidney, brain organoids etc.
- iPS cells treated with particular media in suspension leads to embryoid bodies developing
- Plating on neural induction medium in suspension then differentiation into a Matrigel droplet
- Brain organoid develops
- Stem cell -> embryoid bodies -> neuroectoderm -> expanded neuroepithelium -> cerebral tissue
- Could be used to study developmental pathologies (like autism)
