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.
Wednesday, February 27, 2013
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
Thursday, February 21, 2013
Papers, videos, and transcripts
Hello my Stem Cell Biology people!
First of all, remember to keep checking the google doc of the lecture transcripts. I always update it ASAP and I've been trying to correct my spelling mistakes and add in a few more explanatory words in case I'm not sure the lecturer was clear on something.
Also I'm experimenting with recording the lectures, b/c for some, they are better audio learners. Bear with me as I struggle with technology.
Finally, we have one or two papers assigned for every lecture, so I'll post a sum-up of the papers. I still recommend you read the papers and then read the sum-up, because 1) you need practice if you ever want to be able to digest the condensed jargon of science writing and 2) the more you read the material over and over again the more it sticks in your head.
Here it is from Lecture 1 (Please excuse my casual language. It is my opinion that to make science available to the general, you must speak "layman"...seriously though. I am not trying to condescend to anyone, I just want to make sure people can understand, because that's the foundation to building up knowledge later on.)
Also remember that every paper wants to tell you something, but it is always up to you if you choose to follow or take it for its word. Always ask questions (is this right? did they do this correctly? did they consider this? etc etc) Whatever I write below is not an expression of something I believe in, but just a paraphrase of what I interpreted the paper was trying to say. I'm always open to discussion! (hence, comment box)
Spradling, Allan C. "The living-tissue microscope: the importance of studying stem cells in their natural, undisturbed microenvironment." J Pathol 2011; 225: 161-162.
We need tools to look at cells in their daily lives without disturbing them.
Why? Because we would find out all about normal cell lives, diseased cell lives, new cell to cell and cell to gene and gene to gene pathways that we can't see from frozen or dead cells.
We have the tools to do this:
Small multicell structures with labels can be seen live, for a long period of time (1-3 hr) and at the level of a single cell.
approach referenced from Gaisa et al (see citation 4 in this paper) where they used mitochondrial DNA mutations to trace where each cell came from (lineage analysis)
This is super important for stem cell research.
Why? they are rare and hard to find and hard to identify and basically impossible to label with gene expression tag
But you can do it with lineage analysis!
How did they do it back in the day without being able to creep on cells in vivo?
well, they took tissue from active stem cell tissues (like bone marrow or testis), and put them in a host with little to no stem cell activity, and then voila, cells were being made there.
researchers figured out that there needed to be an area "stem cell niche" to for stem cells to be enriched and function
BUT they couldn't tell if all the cells they transplanted were actually stem cells or just some of them were…
some later studies in flies and mice showed that even daughter cells from stem cells that had begun differentiating (progenitor cells we call them now) could reverse and go back to being stem cell if they found a niche-->hmm, it was now supposed that tissues often contain lots of potential stem cells as well as stem cells.
But we still can't pick out and identify specific actual stem cells until we can look at them in vivo without disturbance. We still don't know where they are in most mammalian tissues.
But we can fix that! We need everyone to do lineage tracing in all of the organs!
it used to be that people searched for stem cells on guesses and unfounded assumptions:
myth: most stem cells are quiet and reproduce only sporadically.
assumption: it must be cells that are still labeled with BrdU (thymidine analog, gets into DNA) after a long time--because they are quiet--must be stem cells.
truth**: at least six types of stem cells have been shown to divide continuously
myth: stem cells spawn huge numbers of daughter cells and these may even be extremely diverse cells (different cell types) once you stick it into tissue culture
assumption: if we stick cells into tissue and they don't suddenly reproduce a crap ton of cells, they must not be stem cells…
truth: most stem cells are maintained as stem cells only by their niche. if they leave or are pushed out, they stop being stem cells and differentiate.
example: mouse intestinal stem cells need specific cytokines and actual niche cells and other stuff in order to successfully propagate.
(**Keep in mind that I only say "truth" to mean that there is evidence to the contrary, not that this is the as-laid-down-by-the-laws-of-nature-factual-truth. Remember always to have a healthy dose of skepticism in science.)
we only figured all that out after the cells were identified by lineage analysis and we could characterize their niche and signals in the niche
unless we have an exact replica of the niche, stem cell in tissue culture will look nothing like stem cells in vivo.
This is applicable to fields outside stem cell research.
embryo cells have constant signaling and interaction with their environment during development
cells in culture also have change in genes/epigenetics, even if their overall karyotype is still the same.
this is all likely due to the fact that they are stressed in an environment not like what they should be in (different signals, treatments, etc)
this is why tissue culture instead of just cell culture is better for multicellular biology
lessons learned from stem cell research:
if we still want to use cell culture but we want to look at things that don't happen just by themselves, we need new tools
we have to look at the events in vivo or in tissue culture
when we figure stuff out about the cells of interest and their environment, then we can create a replica cell culture
then we gotta double-check our model to make sure cells in vitro are behaving just as we had already observed in vivo
you have to follow these steps if you want accurate replication of the crazy complicated system of biology that every cell activity depends on.
good thing new developments in live imaging and lineage analysis are going to make this easier
just remember that you don't want to destroy the very biological events/systems that you wanted to study in the first place.
First of all, remember to keep checking the google doc of the lecture transcripts. I always update it ASAP and I've been trying to correct my spelling mistakes and add in a few more explanatory words in case I'm not sure the lecturer was clear on something.
Also I'm experimenting with recording the lectures, b/c for some, they are better audio learners. Bear with me as I struggle with technology.
Finally, we have one or two papers assigned for every lecture, so I'll post a sum-up of the papers. I still recommend you read the papers and then read the sum-up, because 1) you need practice if you ever want to be able to digest the condensed jargon of science writing and 2) the more you read the material over and over again the more it sticks in your head.
Here it is from Lecture 1 (Please excuse my casual language. It is my opinion that to make science available to the general, you must speak "layman"...seriously though. I am not trying to condescend to anyone, I just want to make sure people can understand, because that's the foundation to building up knowledge later on.)
Also remember that every paper wants to tell you something, but it is always up to you if you choose to follow or take it for its word. Always ask questions (is this right? did they do this correctly? did they consider this? etc etc) Whatever I write below is not an expression of something I believe in, but just a paraphrase of what I interpreted the paper was trying to say. I'm always open to discussion! (hence, comment box)
Spradling, Allan C. "The living-tissue microscope: the importance of studying stem cells in their natural, undisturbed microenvironment." J Pathol 2011; 225: 161-162.
We need tools to look at cells in their daily lives without disturbing them.
Why? Because we would find out all about normal cell lives, diseased cell lives, new cell to cell and cell to gene and gene to gene pathways that we can't see from frozen or dead cells.
We have the tools to do this:
Small multicell structures with labels can be seen live, for a long period of time (1-3 hr) and at the level of a single cell.
approach referenced from Gaisa et al (see citation 4 in this paper) where they used mitochondrial DNA mutations to trace where each cell came from (lineage analysis)
This is super important for stem cell research.
Why? they are rare and hard to find and hard to identify and basically impossible to label with gene expression tag
But you can do it with lineage analysis!
How did they do it back in the day without being able to creep on cells in vivo?
well, they took tissue from active stem cell tissues (like bone marrow or testis), and put them in a host with little to no stem cell activity, and then voila, cells were being made there.
researchers figured out that there needed to be an area "stem cell niche" to for stem cells to be enriched and function
BUT they couldn't tell if all the cells they transplanted were actually stem cells or just some of them were…
some later studies in flies and mice showed that even daughter cells from stem cells that had begun differentiating (progenitor cells we call them now) could reverse and go back to being stem cell if they found a niche-->hmm, it was now supposed that tissues often contain lots of potential stem cells as well as stem cells.
But we still can't pick out and identify specific actual stem cells until we can look at them in vivo without disturbance. We still don't know where they are in most mammalian tissues.
But we can fix that! We need everyone to do lineage tracing in all of the organs!
it used to be that people searched for stem cells on guesses and unfounded assumptions:
myth: most stem cells are quiet and reproduce only sporadically.
assumption: it must be cells that are still labeled with BrdU (thymidine analog, gets into DNA) after a long time--because they are quiet--must be stem cells.
truth**: at least six types of stem cells have been shown to divide continuously
myth: stem cells spawn huge numbers of daughter cells and these may even be extremely diverse cells (different cell types) once you stick it into tissue culture
assumption: if we stick cells into tissue and they don't suddenly reproduce a crap ton of cells, they must not be stem cells…
truth: most stem cells are maintained as stem cells only by their niche. if they leave or are pushed out, they stop being stem cells and differentiate.
example: mouse intestinal stem cells need specific cytokines and actual niche cells and other stuff in order to successfully propagate.
(**Keep in mind that I only say "truth" to mean that there is evidence to the contrary, not that this is the as-laid-down-by-the-laws-of-nature-factual-truth. Remember always to have a healthy dose of skepticism in science.)
we only figured all that out after the cells were identified by lineage analysis and we could characterize their niche and signals in the niche
unless we have an exact replica of the niche, stem cell in tissue culture will look nothing like stem cells in vivo.
This is applicable to fields outside stem cell research.
embryo cells have constant signaling and interaction with their environment during development
cells in culture also have change in genes/epigenetics, even if their overall karyotype is still the same.
this is all likely due to the fact that they are stressed in an environment not like what they should be in (different signals, treatments, etc)
this is why tissue culture instead of just cell culture is better for multicellular biology
lessons learned from stem cell research:
if we still want to use cell culture but we want to look at things that don't happen just by themselves, we need new tools
we have to look at the events in vivo or in tissue culture
when we figure stuff out about the cells of interest and their environment, then we can create a replica cell culture
then we gotta double-check our model to make sure cells in vitro are behaving just as we had already observed in vivo
you have to follow these steps if you want accurate replication of the crazy complicated system of biology that every cell activity depends on.
good thing new developments in live imaging and lineage analysis are going to make this easier
just remember that you don't want to destroy the very biological events/systems that you wanted to study in the first place.
Sunday, February 17, 2013
Cell Biology Study Guides
So I realized I must have shared my Cell Bio exam guides elsewhere or in person, b/c it's not on this blog!
So here they are! Cell Bio exam 1 guide and exam 2 guide. There's already a post for exam 3 somewhere on this blog.
exam 1: https://docs.google.com/file/d/0B99-sSwVe231OWM2YjA3OGItOTU3Ni00MGFiLWEzYzEtMzViNTczMTljYmY5/edit?usp=sharing&authkey=CLKk8NQB
exam 2: https://docs.google.com/file/d/0B99-sSwVe231NTI4Mzk1MWMtMGQwYi00M2JlLWFkM2YtMGRkYTM4NDNhYTFl/edit?usp=sharing&authkey=COCampkH
Again, let me know if the links don't work! (via comment or something)
So here they are! Cell Bio exam 1 guide and exam 2 guide. There's already a post for exam 3 somewhere on this blog.
exam 1: https://docs.google.com/file/d/0B99-sSwVe231OWM2YjA3OGItOTU3Ni00MGFiLWEzYzEtMzViNTczMTljYmY5/edit?usp=sharing&authkey=CLKk8NQB
exam 2: https://docs.google.com/file/d/0B99-sSwVe231NTI4Mzk1MWMtMGQwYi00M2JlLWFkM2YtMGRkYTM4NDNhYTFl/edit?usp=sharing&authkey=COCampkH
Again, let me know if the links don't work! (via comment or something)
Sunday, February 10, 2013
Stem Cells
For the purpose of the class I'm taking, I'll be making three kinds of posts:
1) script -> the class at our university does not have podcasting, so I will be transcribing a misspelling-prone version of near-verbatim of the lectures. Please find it here: https://docs.google.com/document/d/1iFMdbzr9tr0M8PuvGmHvYGXVrPMIAV78OSxW2cGsQxM/edit?usp=sharing
I have little time to spellcheck or grammar check, so please comment if you find something confusing that I can clarify!
2) study guide -> this will be prepared prior to every exam. hopefully it helps!
3) list of papers -> a significant portion of this class involves reading primary documentation, so I will be listing citations of the papers we read (and that will contain info that I will probably include in my study guides).
Ready for stem cells!
1) script -> the class at our university does not have podcasting, so I will be transcribing a misspelling-prone version of near-verbatim of the lectures. Please find it here: https://docs.google.com/document/d/1iFMdbzr9tr0M8PuvGmHvYGXVrPMIAV78OSxW2cGsQxM/edit?usp=sharing
I have little time to spellcheck or grammar check, so please comment if you find something confusing that I can clarify!
2) study guide -> this will be prepared prior to every exam. hopefully it helps!
3) list of papers -> a significant portion of this class involves reading primary documentation, so I will be listing citations of the papers we read (and that will contain info that I will probably include in my study guides).
Ready for stem cells!
Thank you!
Hello! I've been missing for a while (read as "not taking science classes"). This semester I will be back with Stem Cell Biology.
I've just discovered that this blog has rocketed in pageviews over the course of the past several months. I'm not sure what happened, but at least half of my total pageviews have come from Belgium. Not sure who's reading up on science, but I certainly hope it helps! It's very encouraging to know people are looking at what I post, since the purpose of this is to try and share science in a more understandable and explained way than the condensed jargon-dense speech of textbooks.
Cheers!
I've just discovered that this blog has rocketed in pageviews over the course of the past several months. I'm not sure what happened, but at least half of my total pageviews have come from Belgium. Not sure who's reading up on science, but I certainly hope it helps! It's very encouraging to know people are looking at what I post, since the purpose of this is to try and share science in a more understandable and explained way than the condensed jargon-dense speech of textbooks.
Cheers!
Thursday, May 10, 2012
Development Biology Exam 3 (Final but not comprehensive)
http://dl.dropbox.com/u/22080433/3777_001.pdf
http://dl.dropbox.com/u/22080433/3778_001.pdf
http://dl.dropbox.com/u/22080433/4148_001.pdf
http://dl.dropbox.com/u/22080433/4467_001.pdf
And don't forget to check out fly battle vids:
http://www.youtube.com/watch?v=ia57rw7PE_8
http://www.youtube.com/watch?v=4pDU-cqvKJc
and fly sex vids:
http://www.youtube.com/watch?v=zXXqQ2zJVMA
*5/13/12 edit: Thanks to Nadav who pointed out this nuance: stem cells can renew indefinitely, and progenitor cells can also self-renew, but only for a few more rounds.
*5/14/12 edit: More for future sake, it's not unoplakin but uroplakin in 4/13/12 lecture (which makes so much more sense i.e. ureter and all that) AND for 4/18/12 lecture, I wrote in for bone marrow transplantation that one needs mice clones, but actually that's not necessary. Using naked mice means they don't have an immune system to reject transplants anyway. The reason that's difficult in humans is you can't irradiate a human to ablate leukocytes (I mean you could, but who'd let you?)
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