Stem cell biology

Question 1

What is considered the intial breakthrough of stem cell biology and regenerative medicine?

Answer 1

The discovery of multipotent stem cells in mice in 1961

Question 2

What was considered the biggest breakthrough in SCNT?

Answer 2

The cloning of Dolly the sheep in 1997 by Keith Cambell and Ian Wilmut at the University of Edinburgh in Scotland

Question 3

Human ESCs were first isolated by whom, in what year?

Answer 3

Dr. James Thomson, in 1998. Led to the establishment of the H9 line

Question 4

Who first generated iPSCs, and in which organism?

Answer 4

iPSCs were first generated in 2006 in mice using retrovirus, by Takahashi et al

Question 5

Who disoverd VSELS, and how?

Answer 5

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).

Question 6

Who first discovered the existence of PSCs?

Answer 6

Drs James Till and Ernest McCulloh at the University of Toronto, Canada

Question 7

Who discoverd FGF-2 played a role in maintaining hESCs stem cells in the undifferentiated state?

Answer 7

Dvorak, in 2005:

exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling

Question 8

What pathway does FGF-2 activate?

Answer 8

FGF-2 activates FGF receptors leading to activation of the MAPK pathway( which is also activated by insulin-like growth factor receptors and EGFRs)

Question 9

Who discovered Oct4 was the most important reprogramming factor, and how?

Answer 9

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)

Question 10

What are the Yamanaka factors?

Answer 10

“OSKM”
Oct4, SOX2, KLF4- cMyc, identified in 2006- Nanog was dispensable

Question 11

Describe the assay system Yamanaka used to narrow down the 24 candidate pluripotency genes to 4

Answer 11

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

Question 12

What are the two defining properties of hESCs?

Answer 12

1) The ability to self-renew
* The ability to sustain proliferation indefinitely

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2) Pluripotency
* The ability to differentiate into the three different germ layers( ectoderm, mesoderm, endoderm)

Question 13

What are the five basic categories of stem cells?

Answer 13

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

Question 14

ESCs and iPSCs show great promise in which four fields?

Answer 14

1) Regenerative Medicine
2) Disease Modeling
3) Drug discovery screening
4) Human developmental biology

Question 15

What is the most stringent way to assess the pluripotency of iPSCs?

Answer 15

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)

Question 16

How would one assess the pluripotency of iPSCs in vitro?

Answer 16

Embryoid body formation

Question 17

In a teratoma formation assay or embryoid body formation assay, which markers could be used to assess lineage commitment to ectoderm, mesoderm, and endoderm?

Answer 17

Ectoderm: PAX-6 and SOX2

Endoderm: CSCR4 and SOX17

Mesoderm: SM22a and CD144

Question 18

How large are VSELS?

Answer 18

5 to 7 um in humans(slightly smaller than RBCs)

Question 19

Which genes to VSELs express?

Answer 19

nuclear Oct-4A, SSEA, Nanog, Rex1

Question 20

In January 2018, Chinese scientists used what kind of cells to clone which type of organism?

Answer 20

They used fetal fibroblasts to clone two female macaques, thus creating the first primates to be cloned by SCNT

Question 21

Why would SCNT-cloned primates be useful for biomedical applications?

Answer 21

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.

Question 22

What are some common iPSC parent cell sources?

Answer 22

Blood, skin, urine

Question 23

Why do hiPSCs offer potential for personalized medicine?

Answer 23

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

Question 24

What three features should be considered when selecting a somatic cell source for reprogramming

Answer 24

1) Non-invasive
2) Easily accessible
3) Reproducible

Question 25

The survival, quiesence, and activation of adult stem cells is thought to depend on what?

Answer 25

Factors from the microenvironment

Question 26

What is the major drawback of retroviral/ lentiviral reprogramming strategies?

Answer 26

They integrate into the genome, which could disrupt endogenous gene expression and is associated with the risk of insertional mutagenesis.

Question 27

What is the most important type of adult stem cell?

Answer 27

MSCs

Question 28

What are the major sources of MSCs in vivo?

Answer 28

cord blood, bone marrow, adipose tissue, amniotic fluid and menstrual blood

Question 29

What is the “second generation” of stem cell re-programming techniques?

Answer 29

Direct cellular reprogramming in vivo:

leveraging the native microenvironment to obtain natural products and obtain in situ recovery of locally damaged tissues

Question 30

in vivo somatic re-programming has made substantial advances in converting which two cell types?

Answer 30

cardiomyocyte and neuronal cell fates

Question 31

What is thought to be responsible for the lineage bias of certain iPSc lines?

Answer 31

Epigenetic memory: iPSCs retain an epigenetic memory of being the parent cell

Question 32

What is the difference between naive and primed iPSCs?

Answer 32

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.

Question 33

Which factors can serve as substitutes for c-Myc and KLF4 during reprogramming?

Answer 33

Nanog and Lin-28

Question 34

List the four techniques to reprogram somatic cells?

Answer 34

1) Integrating/ Viral
2) Non-integrating Viral
3) Self-excising vectors
4) Non-integrating/ non-viral

Question 35

Give examples of integrating/viral reprogramming

Answer 35

Using lentivirus/ Retrovirus to deliver reprogramming factors

This strategy is highly efficient, but not deemed clinically safe due to possibility of insertional mutagenesis

Question 36

What is an example of non-integrating viral reprogramming?

Answer 36

Sendai virus, adeno virus, episomal vectors, mRNA/cDNA plasmids

Question 37

What is an example of reprogramming using self excising vectors?

Answer 37

Cre-Lox/ piggy bac transposon

The biggest benefit here is that the the transgene is not integrating

Question 38

What is an example of non-integrating, non-viral reprogramming strategy?

Answer 38

using small molecules, such as valproic acid, chir99021, micro RNA to induce cell fate change

Question 39

What are three major questions that will be need to be adressed when pursuing de-differentiation as a regenerative strategy?

Answer 39

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?