Stem cell biology Flashcards
What is considered the intial breakthrough of stem cell biology and regenerative medicine?
The discovery of multipotent stem cells in mice in 1961
What was considered the biggest breakthrough in SCNT?
The cloning of Dolly the sheep in 1997 by Keith Cambell and Ian Wilmut at the University of Edinburgh in Scotland
Human ESCs were first isolated by whom, in what year?
Dr. James Thomson, in 1998. Led to the establishment of the H9 line
Who first generated iPSCs, and in which organism?
iPSCs were first generated in 2006 in mice using retrovirus, by Takahashi et al
Who disoverd VSELS, and how?
VSELs were first discovered in 2006, by Ratajczak-
The group identified in bone marrow and other adult tissues a population of very small embryonic like stem cells (VSELs) which express several markers characteristic for pluripotent stem cells (PSC) that are characteristic for epiblast/germ line- derived stem cells(i.e. Oct4).
Who first discovered the existence of PSCs?
Drs James Till and Ernest McCulloh at the University of Toronto, Canada
Who discoverd FGF-2 played a role in maintaining hESCs stem cells in the undifferentiated state?
Dvorak, in 2005:
exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling
What pathway does FGF-2 activate?
FGF-2 activates FGF receptors leading to activation of the MAPK pathway( which is also activated by insulin-like growth factor receptors and EGFRs)
Who discovered Oct4 was the most important reprogramming factor, and how?
Oct4 was found to be required for the formation of the naïve epiblast, because the inner cell mass of Oct4-null embryos lacks pluripotent characteristics(mouse model, Nichols 1998)
What are the Yamanaka factors?
“OSKM”
Oct4, SOX2, KLF4- cMyc, identified in 2006- Nanog was dispensable
Describe the assay system Yamanaka used to narrow down the 24 candidate pluripotency genes to 4
developed an assay system in which the induction of the pluripotent state could be detected as resistance to G418. They inserted a βgeo cassette (a fusion of the β-galactosidase and neomycin resistance genes) into the mouse Fbx15 gene by homologous recombination. Although specifically expressed in mouse ES cells and early embryos, Fbx15 is dispensable for the maintenance of pluripotency and mouse development. ES cells homozygous for the βgeo knockin construct (Fbx15βgeo/βgeo) were resistant to extremely high concentrations of G418
What are the two defining properties of hESCs?
1) The ability to self-renew
* The ability to sustain proliferation indefinitely
2) Pluripotency
* The ability to differentiate into the three different germ layers( ectoderm, mesoderm, endoderm)
What are the five basic categories of stem cells?
1) Embryonic Stem Cells
2) VSELS( very small emryonic like stem cells)
3) Nuclear Transfer stem cells (NTSCs)
4) Reprogrammed Stem cells
5) Adult stem cells
ESCs and iPSCs show great promise in which four fields?
1) Regenerative Medicine
2) Disease Modeling
3) Drug discovery screening
4) Human developmental biology
What is the most stringent way to assess the pluripotency of iPSCs?
The most stringent way is to perform a teratoma test- iPSCs are injected into an immun compromised animal, and evaluated for the emergence of all three germ layers(ectoderm, endoderm, mesoderm)
How would one assess the pluripotency of iPSCs in vitro?
Embryoid body formation
In a teratoma formation assay or embryoid body formation assay, which markers could be used to assess lineage commitment to ectoderm, mesoderm, and endoderm?
Ectoderm: PAX-6 and SOX2
Endoderm: CSCR4 and SOX17
Mesoderm: SM22a and CD144
How large are VSELS?
5 to 7 um in humans(slightly smaller than RBCs)
Which genes to VSELs express?
nuclear Oct-4A, SSEA, Nanog, Rex1
In January 2018, Chinese scientists used what kind of cells to clone which type of organism?
They used fetal fibroblasts to clone two female macaques, thus creating the first primates to be cloned by SCNT
Why would SCNT-cloned primates be useful for biomedical applications?
If combined with CRISPR-Cas9 gene editing technology, it could be used to create genetically uniform primate lines, which would be useful for mechanistic studies of disease, since genetic variation is accounted for.
What are some common iPSC parent cell sources?
Blood, skin, urine
Why do hiPSCs offer potential for personalized medicine?
Since they are autologously derived, they avoid the potential of immune rejection(which is an issue for hESCs)
They also avoid the ethics controversy surrounding ESCs, because they are derived from adult somatic cells
What three features should be considered when selecting a somatic cell source for reprogramming
1) Non-invasive
2) Easily accessible
3) Reproducible
The survival, quiesence, and activation of adult stem cells is thought to depend on what?
Factors from the microenvironment
What is the major drawback of retroviral/ lentiviral reprogramming strategies?
They integrate into the genome, which could disrupt endogenous gene expression and is associated with the risk of insertional mutagenesis.
What is the most important type of adult stem cell?
MSCs
What are the major sources of MSCs in vivo?
cord blood, bone marrow, adipose tissue, amniotic fluid and menstrual blood
What is the “second generation” of stem cell re-programming techniques?
Direct cellular reprogramming in vivo:
leveraging the native microenvironment to obtain natural products and obtain in situ recovery of locally damaged tissues
in vivo somatic re-programming has made substantial advances in converting which two cell types?
cardiomyocyte and neuronal cell fates
What is thought to be responsible for the lineage bias of certain iPSc lines?
Epigenetic memory: iPSCs retain an epigenetic memory of being the parent cell
What is the difference between naive and primed iPSCs?
Both are states of pluripotency, but exhibit slightly different properties. The naïve state represents the cellular state of the preimplantation mouse blastocyst inner cell mass, while the primed state is representative of the post-implantation epiblast cells. These two cell types exhibit clearly distinct developmental potential, as evidenced by the fact that naïve cells are able to contribute to blastocyst chimeras, while primed cells cannot.
Which factors can serve as substitutes for c-Myc and KLF4 during reprogramming?
Nanog and Lin-28
List the four techniques to reprogram somatic cells?
1) Integrating/ Viral
2) Non-integrating Viral
3) Self-excising vectors
4) Non-integrating/ non-viral
Give examples of integrating/viral reprogramming
Using lentivirus/ Retrovirus to deliver reprogramming factors
This strategy is highly efficient, but not deemed clinically safe due to possibility of insertional mutagenesis
What is an example of non-integrating viral reprogramming?
Sendai virus, adeno virus, episomal vectors, mRNA/cDNA plasmids
What is an example of reprogramming using self excising vectors?
Cre-Lox/ piggy bac transposon
The biggest benefit here is that the the transgene is not integrating
What is an example of non-integrating, non-viral reprogramming strategy?
using small molecules, such as valproic acid, chir99021, micro RNA to induce cell fate change
What are three major questions that will be need to be adressed when pursuing de-differentiation as a regenerative strategy?
1) How far should cells be reversed along de-differentiation?
2) What percentage of cells should be induced to de-differentiate?
3) When is the best timing induce dedifferentiation after injury?
