So here I have attempted to simplify by just taking an audio recording of the lecture by Dr. Barry Zirkin (this time on Leydig stem cells). I have to forewarn you that my computer's Audacity program also succinctly captured the sound of my typing (as I transcribe the lecture as well). Apologies if that annoys you :(. Still, it captures the lecture all the same.
https://dl.dropbox.com/u/22080433/StemCell2013Lecture5.mp3
Also I still update the transcribed lectures on the google doc (see link some posts earlier) and in two weeks, we have our first exam, so in one week, look out for some study guides! :D
Wednesday, February 27, 2013
Lecture 2 Paper Sumup: induced Pluripotent Stem Cells
Takahashi K and Yamanaka S. "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors." Cell 126, 2006: 663-676.
Introduction:
ES cells are pluripotent: they come from mammalian embryo and can make cells from all 3 germ layers.
- super useful for treating diseases
- but ethically controversial (can we use human embryos for this purpose?)
- and physically difficult if implanted tissue is rejected by host
possible solution? get pluripotent cells from host themselves!
- take normal body cell (somatic cell) DNA and stick it in an oocyte or fuse the cell with an ES cell
- the other cell contents of ES cells/oocyte have been shown to contain some factors that make somatic cells pluripotent!
- we know some factors that maintain pluripotency, but maybe these factors also INDUCE pluripotency
what do we know about factors that maintain pluripotency:
- Oct3/4, Nanog handle maintenance
- Stat3, E-Ras, c-myc, Klf4, Beta-catenin are highly expressed in tumors--they also help maintain long term ES cell phenotype and proliferation
Results:
We tried out 24 genes that could have been the factors that induce pluripotency.
- experiment: if gene X induces pluripotency, cell will be resistant to G418 (a molecule that inhibits protein synthesis).
- ergo, if we see cell resistant to G418, then the gene that we unregulated in the cell is a factor that induces pluripotency.
step 1: in mouse, knock out the gene Fbx15. This gene is super important for maintaining pluripotency in mouse development.
- ES cell with knocked out Fbx15 resist G418
- somatic cells with knocked out Fbx15 cannot resist G418
step 2: one by one, insert each of the 24 candidate genes into these knockout embryonic mice cells
- no resistance observed
- ergo, these genes cannot induce pluripotency by themselves
step 3: upregulate all 24 genes in these knockout cells
- get lots of resistant colonies!
- some of these look very similar to ES cells (morphology, proliferation traits, gene expression markers, etc)
step 4: upregulate all but 1 gene in these knockout cells
- found 10 genes that, once you did NOT upregulate them in the cells, you did not get resistant colonies
- ergo, these are super important factors that you can't NOT put in in order to induce pluripotency
step 5: upregulate all 10 special genes in knockout cells
- get lots of resistant colonies!
- many of these look similar to ES cells
step 6: upregulate all except 1 of these 10 special genes in knockout cells
- not including either Oct3/4 or Klf4 resulted in no resistant colonies
- not including Sox2 resulted in very very few resistant colonies
- not including c-myc resulted in weird looking resistant colonies
- not including any of the others produced all resistant colonies, so the others were not as important as the above 4.
step 7: upregulate only those 4 super special genes in knockout cells
- get same result as step 5
- culture and confirm that these are iPSC (induced pluripotent stem cells)
step 8: upregulate pairs and triplets of these 4 super special genes in knockout cells
- no two of them could form any resistant colonies
- 2 triplets produced a few colonies but they did not survive further culturing
- 2 other triplets produced more colonies but looked weird (different from ES or previously determined iPSC)
step 9: do gene expression analysis of iPSCs induced with the various combos
- the 4 combo and the 10 combo cells, both are similar to ES cell expression profiles but not exactly the same
- the 3 combo cells were very very different
step 10: try to make teratomas with these various combo-formed iPSCs
- there was inconsistent data: some of the 10 combos and the 4 combos made teratomas with cell types of all 3 layers, but some of the same combos could only form 2 or 1 of the germ layers
- so conclude the majority of the 10-combo and 4-combo cells are pluripotent, but not all
- tumors from 3-combo cells did not differentiate = not pluripotent!
- similar results when trying to form embryoid bodies in culture as opposed to forming teratomas in vivo.
step 11: introduce the 4 combo gene into mouse tail fibroblasts (somatic cells) and then inject these cells into blastocyst
- were able to observe that these injected cells helped form some of the germ layers and baby mice were actually born from these blastocysts that received injections!
step 12: compare gene expression levels of these 4 factors with protein expression levels btw iPSC and ES
- saw that while some of these genes were higher or lower in iPSC cells than in normal ES cells, the protein levels (Western blot!) were about the same!
step 13: try to grow iPSC without them differentiating in culture
- they always differentiated unless they were provided "feeder cells" in the same culture
Discussion:
- Oct3/4, Sox2, Nanog are essential for maintaining pluripotency
- Oct 3/4 and Sox 2 are essential for MAKING iPSCs
- Nanog is not important for that
- c-Myc Klf4 are also essential
- c-Myc upregulates genes for proliferation and transformation
- it affects some histone modifying enzymes (histone acetyltransferase, for example)
- there are a LOT (upt to 25000) of sites for c-Myc binding in mammal genome
- this is way more than what we'd guess for Oct3/4 or Sox2 binding sites
- it could be that c-Myc causes global histone acetylation, causing a lot of the genome to open up, so that Oct3/4 and Sox2 can find all their target binding sites
- what about Klf4? represses p53
- okay, what does p53 do? It suppresses Nanog during differentiation
- so if you repress p53, you enable Nanog, which should normally NOT be active for differentiation.
- this might contribute to making the iPSC or at least ES-like cell phenotype
- Klf4 activates p21 which suppresses proliferation, and c-Myc suppresses p21. This opposites relationship of c-Myc and Klf4 might be important (in other words, we're just guessing)
- one important question: which cells of the tissue given these four factors are becoming iPSCs?
- only a small portion of cells treated with the 4 factors become iPSCs
- maybe it's the progenitor/stem cells that already exist in tissue that are kinda multipotent but not pluripotent that transform into pluripotent cells
- the frequency doesn't change when we try this out with bone marrow, which should have a high percentage of progenitor/stem cells to begin with to change
- so it can't be those cells..
- maybe getting the right expression level of each factor in the cells is important
- experimental evidence: just a 50% increase or decrease in Oct3/4 proteins in an ES cell causes it to differentiate and lose pluripotency
- we know our iPSC clones overexpress RNA levels but their protein levels of the 4 factors are just right
- but these cells must be able to regulate that, b/c high high levels are necessary to become ES-cells but in order to stay ES-like, too much of the 4 factors is badddd
- they might need some chromosomal alterations too to stay ES-like
- this may be spontaneous or induced by some of the 4 factors
- where the retrovirus brings in the transgenes to overexpress the 4 factors also matters: could have impacted the expression of any native genes depending on how the transgene got shoved into the genome
- another question: are these 4 factors also important when we're trying to reprogram somatic cells by fusion with ES cells or plucking out nuclei and putting them in oocytes?
- the precise roles of Klf4 and c-Myc are also confusing and vague. they aren't essential for mouse development before the egg implants. c-Myc isn't detectable in oocytes at all. Hmm??
- well, related proteins, L-myc and Klf17 and Klf7 do exist. maybe Klf4 and C-myc's real properties are being supplanted by these relatives in wildtype development
- some other questions that this paper brings up...
- still unsure if these 4 factors can make pluripotent cells out of human somatic cells.
- testing/experimental process is going to require super specific culture environments
- but this is all really cool in the search for the tools to control pluripotency, and one day we might be able to make pluripotent cells from a patient's somatic cells.
Introduction:
ES cells are pluripotent: they come from mammalian embryo and can make cells from all 3 germ layers.
- super useful for treating diseases
- but ethically controversial (can we use human embryos for this purpose?)
- and physically difficult if implanted tissue is rejected by host
possible solution? get pluripotent cells from host themselves!
- take normal body cell (somatic cell) DNA and stick it in an oocyte or fuse the cell with an ES cell
- the other cell contents of ES cells/oocyte have been shown to contain some factors that make somatic cells pluripotent!
- we know some factors that maintain pluripotency, but maybe these factors also INDUCE pluripotency
what do we know about factors that maintain pluripotency:
- Oct3/4, Nanog handle maintenance
- Stat3, E-Ras, c-myc, Klf4, Beta-catenin are highly expressed in tumors--they also help maintain long term ES cell phenotype and proliferation
Results:
We tried out 24 genes that could have been the factors that induce pluripotency.
- experiment: if gene X induces pluripotency, cell will be resistant to G418 (a molecule that inhibits protein synthesis).
- ergo, if we see cell resistant to G418, then the gene that we unregulated in the cell is a factor that induces pluripotency.
step 1: in mouse, knock out the gene Fbx15. This gene is super important for maintaining pluripotency in mouse development.
- ES cell with knocked out Fbx15 resist G418
- somatic cells with knocked out Fbx15 cannot resist G418
step 2: one by one, insert each of the 24 candidate genes into these knockout embryonic mice cells
- no resistance observed
- ergo, these genes cannot induce pluripotency by themselves
step 3: upregulate all 24 genes in these knockout cells
- get lots of resistant colonies!
- some of these look very similar to ES cells (morphology, proliferation traits, gene expression markers, etc)
step 4: upregulate all but 1 gene in these knockout cells
- found 10 genes that, once you did NOT upregulate them in the cells, you did not get resistant colonies
- ergo, these are super important factors that you can't NOT put in in order to induce pluripotency
step 5: upregulate all 10 special genes in knockout cells
- get lots of resistant colonies!
- many of these look similar to ES cells
step 6: upregulate all except 1 of these 10 special genes in knockout cells
- not including either Oct3/4 or Klf4 resulted in no resistant colonies
- not including Sox2 resulted in very very few resistant colonies
- not including c-myc resulted in weird looking resistant colonies
- not including any of the others produced all resistant colonies, so the others were not as important as the above 4.
step 7: upregulate only those 4 super special genes in knockout cells
- get same result as step 5
- culture and confirm that these are iPSC (induced pluripotent stem cells)
step 8: upregulate pairs and triplets of these 4 super special genes in knockout cells
- no two of them could form any resistant colonies
- 2 triplets produced a few colonies but they did not survive further culturing
- 2 other triplets produced more colonies but looked weird (different from ES or previously determined iPSC)
step 9: do gene expression analysis of iPSCs induced with the various combos
- the 4 combo and the 10 combo cells, both are similar to ES cell expression profiles but not exactly the same
- the 3 combo cells were very very different
step 10: try to make teratomas with these various combo-formed iPSCs
- there was inconsistent data: some of the 10 combos and the 4 combos made teratomas with cell types of all 3 layers, but some of the same combos could only form 2 or 1 of the germ layers
- so conclude the majority of the 10-combo and 4-combo cells are pluripotent, but not all
- tumors from 3-combo cells did not differentiate = not pluripotent!
- similar results when trying to form embryoid bodies in culture as opposed to forming teratomas in vivo.
step 11: introduce the 4 combo gene into mouse tail fibroblasts (somatic cells) and then inject these cells into blastocyst
- were able to observe that these injected cells helped form some of the germ layers and baby mice were actually born from these blastocysts that received injections!
step 12: compare gene expression levels of these 4 factors with protein expression levels btw iPSC and ES
- saw that while some of these genes were higher or lower in iPSC cells than in normal ES cells, the protein levels (Western blot!) were about the same!
step 13: try to grow iPSC without them differentiating in culture
- they always differentiated unless they were provided "feeder cells" in the same culture
Discussion:
- Oct3/4, Sox2, Nanog are essential for maintaining pluripotency
- Oct 3/4 and Sox 2 are essential for MAKING iPSCs
- Nanog is not important for that
- c-Myc Klf4 are also essential
- c-Myc upregulates genes for proliferation and transformation
- it affects some histone modifying enzymes (histone acetyltransferase, for example)
- there are a LOT (upt to 25000) of sites for c-Myc binding in mammal genome
- this is way more than what we'd guess for Oct3/4 or Sox2 binding sites
- it could be that c-Myc causes global histone acetylation, causing a lot of the genome to open up, so that Oct3/4 and Sox2 can find all their target binding sites
- what about Klf4? represses p53
- okay, what does p53 do? It suppresses Nanog during differentiation
- so if you repress p53, you enable Nanog, which should normally NOT be active for differentiation.
- this might contribute to making the iPSC or at least ES-like cell phenotype
- Klf4 activates p21 which suppresses proliferation, and c-Myc suppresses p21. This opposites relationship of c-Myc and Klf4 might be important (in other words, we're just guessing)
- one important question: which cells of the tissue given these four factors are becoming iPSCs?
- only a small portion of cells treated with the 4 factors become iPSCs
- maybe it's the progenitor/stem cells that already exist in tissue that are kinda multipotent but not pluripotent that transform into pluripotent cells
- the frequency doesn't change when we try this out with bone marrow, which should have a high percentage of progenitor/stem cells to begin with to change
- so it can't be those cells..
- maybe getting the right expression level of each factor in the cells is important
- experimental evidence: just a 50% increase or decrease in Oct3/4 proteins in an ES cell causes it to differentiate and lose pluripotency
- we know our iPSC clones overexpress RNA levels but their protein levels of the 4 factors are just right
- but these cells must be able to regulate that, b/c high high levels are necessary to become ES-cells but in order to stay ES-like, too much of the 4 factors is badddd
- they might need some chromosomal alterations too to stay ES-like
- this may be spontaneous or induced by some of the 4 factors
- where the retrovirus brings in the transgenes to overexpress the 4 factors also matters: could have impacted the expression of any native genes depending on how the transgene got shoved into the genome
- another question: are these 4 factors also important when we're trying to reprogram somatic cells by fusion with ES cells or plucking out nuclei and putting them in oocytes?
- the precise roles of Klf4 and c-Myc are also confusing and vague. they aren't essential for mouse development before the egg implants. c-Myc isn't detectable in oocytes at all. Hmm??
- well, related proteins, L-myc and Klf17 and Klf7 do exist. maybe Klf4 and C-myc's real properties are being supplanted by these relatives in wildtype development
- some other questions that this paper brings up...
- still unsure if these 4 factors can make pluripotent cells out of human somatic cells.
- testing/experimental process is going to require super specific culture environments
- but this is all really cool in the search for the tools to control pluripotency, and one day we might be able to make pluripotent cells from a patient's somatic cells.
Sunday, February 24, 2013
Stem Cells: Lecture 4 Video
Here is my first attempt at recording. I did it less for the purpose of showing slides (because we get that provided at our school) and more for the purpose of audio learners, who don't do well just reading the transcripts of the lectures. I might just try an audio recording instead of video and audio next time.
This is the lecture by Dr. William Wright on the subject of stem spermatogonia.
https://www.youtube.com/watch?v=br7STDDbx24
This is the lecture by Dr. William Wright on the subject of stem spermatogonia.
https://www.youtube.com/watch?v=br7STDDbx24
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