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Exam 1 Flashcards

1
Q

Process of embryonic stem cell isolation

A
  1. Frozen blastocysts from fertility clinics
  2. ICM separated from trophoblast (placenta)
  3. Cell are disassociated and cultured
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2
Q

What is the ICM?
What day post fertilization do we see pluripotent stem cells?

A

Cluster of cells that have the potential to become embryos
10-12 days

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3
Q

Sketch diagram on a blastocyst and label ICM

A
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4
Q

Explain the various potencies of cells in mammals and give an example

A
  1. Totipotency - cells have potential to become all things (zygote)
  2. Pluripotency - cells have the potential to become many cells (embryonic stem cells)
  3. Multi potency - cells have the potential to become many things but not all (adult stem cells)
  4. Unipotency - somatic cells (skin cell)
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5
Q

4 common types of stem cells

A
  1. Embryonic
  2. Tissue-specific
  3. Induced pluripotent
  4. Mesenchymal
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6
Q

How are ESC different than iPSCs?

A

ESC are found in the ICM & iPSCs are derived from skin or blood cells

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7
Q

5 sources of Multipotent Stem Cells in the body

A
  1. Bone marrow
  2. Adipose fat
  3. Placental
  4. Hair follicle
  5. Dental pulp
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8
Q

4 areas that stem cells have a positive impact in the future

A
  1. Bone marrow for Leukemia
  2. Nerve cells for parkinsons
  3. Heart muscle for heart disease
  4. Toxicity testing
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9
Q

Name 4 reprogramming factors for mice and additional 2 for humans

A
  1. Sox2
  2. Oct4
  3. cMyc
  4. Klf4
  5. Lin28
  6. Nanog
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10
Q

What is karyotyping? Why is it important?

A

Visual chromosomes in a sample of cells
Helps identify any genetic abnormalities

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11
Q

3 germ layers and example of each

A
  1. Ectoderm - skin cells
  2. Endoderm - organ cells
  3. Mesoderm - connective tissue
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12
Q

Why is 3D cell culture important?

A

it allows for a more similar environment like in vivo
importance: drugs cant penetrate the cell in a 2D state

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13
Q

2 challenges with using stem cells in clinical trials

A

Hit & miss - potential it wouldnt work
Contamination of viruses, bacteria, fungi, yeast

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14
Q

How do you make a human/monkey chimera?

A
  1. Take iPSCs from human and add to monkeys blastocyst (ICM) - both cells are present
  2. Development happens in vitro
  3. Human cells give rise to ecto, endo, mesoderm
  4. Human derived organs in monkey
  5. Kill monkey to retrieve organs
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15
Q

Sources that one can obtain ESCs

A

Spontaneous non elective abortions
in-vitro fertilizations

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16
Q

4 causes that was used to isolate fetal stem cells upon parental consent

A
  1. ectopic pregnancy
  2. threatened abortion
  3. premature rupture of membranes
  4. inevitable abortions
17
Q

Explain the process of IVF

A
  1. mother gets injected for ovarian hyper stimulation
  2. 10-12 days for egg retrieval
  3. sperm prep and incubated with egg
  4. embryo culture (6-8 days)
  5. 6-8 embryos back to uterus & hopefully pregnancy
18
Q

What is SCNT? Explain how Dolly were produced.

A

Somatic Cell Nuclear Transfer

  1. retrieve unfertilized egg from mother and remove nucleus
  2. get somatic skin cell from what you want to clone and use nucleus (DNA) on unfertilized egg with no nucleus
  3. transferred to surrogate mother
  4. blastocyst/embryo forms during gestation period
19
Q

4 strategies to induce epigenetic reprogramming

A

Nuclear Transfer
Cell fusion
Cell explantation
Transduction of reprogramming factors (lenti-viral)

20
Q

2 promises and 2 challenges for Nuclear Transfer and Cell Fusion (epigenetic reprogramming)

A

Nuclear Transfer (promise: reproductive cloning) (challenge: clone is abnormal)

Cell fusion (promise: customized ES cells) (challenge: inefficient, ethics)

21
Q

How to generate a chimera? Draw diagram

A

Human iPSCs into animal blastocysts (nucleated)
Animals will be surrogate and create half/half species

22
Q

OCT4 + SOX2 = ?

A

Activate for pluripotency

23
Q

OCT4 + SOX17 = ?

A

Activate differentiation (endoderm)

24
Q

Describe 4 properties of pluripotent stem cells

A
  1. self renewal, immortal
  2. asymmetric division
  3. potential to become many cells in the adult body
  4. can derive to ectoderm, endoderm, and mesoderm
25
2 advantages and 2 disadvantages of IVF
Advantages: dont need a partner to reproduce, helps for infertility disadvantages: low efficiency rate, high chance of miscarriages, expensive
26
How does the immediate micro environment play a role in reprogramming?
Signal transduction send signals to chromatin regulators, miRNA, and target genes which can activate differentiation and pluripotency genes - change in pH,, nitrogen, neighboring cells (adipose fat, muscle, etc.)
27
Draw timeline required to generate and characterize human iPSCs from skin or blood stem cells
day 0: add virus day 6-7: take out extra virus (non-integrated) change media everyday for 40 days day 30: you will see clones day 30-90: culture and grow ~6 months: characterization of clones
28
What are the 3 core interacting TFs in ESCs/iPSCs and 4 molecular changes performed.
NANOg - stops embryonic ectoderm (skin/brain) OCT4 - binds & shuts down txn factor BMP4 (bone morphogenetic protein) SOX2 - shuts down primitive streak - stops neural/muscle and mesoderm -RPol2 can bind to promoter region of core - transcribes to protein (mRNA - ss of nanog, oct4, sox2) - translated to protein (protein of nanog, oct4, sox2) - protein binds to target gene of pluripotency in nucleus
29
Explain how core TFs interact to induce pluripotency genes and repress differentiation genes
core transcription are activated = active pluripotency inactive = differentiation
30
2 advantages/disadvantages for making iPSCs using the following: - HIV based lenti virus - Moloney based retro viruses - Transient expression of plasmids - Adenovirus
- HIV based lenti virus A: constantly compressed, temporal control over factor expression D: risk of insertional mutagenesis, possible leaky expression - Moloney based retro viruses A: silenced in pluripotent cells, self-silencing eliminates need for timed factor withdrawal D: genomic integration, limited to dividing cells - Transient expression of plasmids A: no viral components, low freq. of genomic integration D: multiple rounds of transinfection are required, delayed kinetics of reprogramming - Adenovirus A: low freq. of genomic integration, easily approved of FDA D: delayed kinetics of reprogramming, repeated infection required for certain cells
31
Explain how core stem cells interact with BMP4 and lineages suppressed/activated by these proteins (DRAW DIAGRAM)
- OCT4 regulates BMP4 - Nanog inhibits embryonic ectoderm Nanog +Oct4 represses the embryonic ectoderm. Oct 4 inhibits the extra-embryonic ectoderm, endoderm. - Sox2 will repress the primitive streak (meso/endoderm) - Oct4 and Sox2 can induce the primitive streak and BMP4 will induce the primitive streak.
32
Concentration fluctuations of Oct4 and Sox2 can induce differentiation to what lineages?
Various concentration fluctuations affect the ES cell differentiation efficiency towards mesoderm and neuroderm - too much Oct4 = differentiate to neuroectoderm + mesendoderm - too much Sox2 = differentiate to neuroectoderm
33
Briefly explain how p300 (co-activator) and STAT3 helps in maintaining pluripotency/differentiation
Nanog, oct4, sox2 are bound together by p300 (enhancer). Maintains pluripotency by activating gene expression, if not they differentiate FOXD3 is for differentiation STAT3 is essential for self-renewal but at late development (adult stem)
34
Other cellular processes are required to maintain potency in ESCs/iPSCs in most mammalian species. Mention any 4 of these.
- Transcription - Post transcriptional regulation - bioenergetics - epigenetics
35
What happens to PGRN (pluripotency gene regulatory network) with changes in energy availability inside the cell?
Low = stable and pluripotent High= core breaks down and differentiates
36
What is quiescence? What is its importance in ESCs and adult stem cells?
Phenomenon in stem cells that are inactive but signal will cause to renew/ differentiate
37
Explain in detail 4 tests required to detect reprogramming with diagrams
1. Invitro Differentiation -induced in cultured cells & are assayed for expression 2. Teratoma Formation -tumors demonstrating the potential to generate differentiated cells 3. Chimera Formation - contribution of cells to normal development following injection into blastocyst 4. Germ Line Contribution - ability to test cells to generate functional germ cells

Stem Cell (3 decks)

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