it was very nice outside this evening.
Monday, October 20, 2008
Saturday, October 18, 2008
take me to your leader
"Where's the kaboom? There was supposed to be an Earth-shattering kaboom!"
- Marvin the Martian
Friday, October 17, 2008
abstract for my research proposal
i'm sure anyone who's reading this is thinking "just what is lilly going to be working on for the next X years (assuming she passes her comps)?" well, here for your reading pleasure is the abstract for my proposal. it's a little long and wordy (like i tend to be), and i really had to edit the hell out of it to fit it into one page in 11 point (or larger) font, but here it is:
Optimization of Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes for Use in Repair of Myocardial Infarction
While embryonic stem cells possess the ability to become virtually any cell type, many of the specific events involved in differentiation are unknown. The ability to direct the differentiation of stem cells into specific lineages would provide scientists with a powerful tool to generate cells for the treatment of many diseases and injuries. Stem cell-based treatment for myocardial infarction is particularly attractive due to the irreversible nature of the injury and the limited inherent regenerative capability of the heart. However transplant of undifferentiated stem cells is risky due to the possibility of teratoma formation, and the ability of these cells to engraft and participate in heart function is unknown. Researchers are beginning to understand the vital steps in cardiomyogenesis, and some of the earliest surface receptors and transcription factors expressed in cardiomyocyte progenitors have recently been identified. We hypothesize that by sorting murine embryonic stem (mES) cells differentiated via embryoid body (EB) formation based on expression of markers specific to the cardiac lineage, and ectopically expressing known cardiac transcription factors in these sorted cells using HSV vectors, we can produce a population of cells highly enriched for cardiomyocytes.
GATA4 and Nkx2.5 are two of the earliest transcription factors expressed in cardiac progenitors. They act together to regulate expression of cardiac specific genes in the developing heart, and exogenous expression of these two factors in mesenchymal stem cells has been shown to drive them towards a cardiac fate. We plan to construct HSV-1 vectors to express these genes in mES and embryoid body cells. HSV vectors are well suited for this work because they provide a pulse of gene expression which mimics the transient expression of developmentally regulated genes. The replication defective HSV vector JDββ has been shown to be capable of transgene expression in these cell types, and has a very low toxicity due to deletion of the immediate early genes ICP4 and ICP22, and reduced expression of ICP0 and ICP27, which are under control of an early (β) promoter. Previous work has also shown that this vector does not interfere with self-renewal or EB formation of mES cells, and does not alter expression of the mesoderm marker Brachyury (Bry). The latter is particularly important for our current work as cardiomyocytes are derived from this Bry positive mesoderm population.
Researchers have engineered a mES cell line which expresses EGFP under the control of the Bry promoter. They used FACS to sort cells based on expression of Bry along with Flk-1 to enrich for cardiac progenitors. A separate group used bioinformatic dissection to identify overexpression of the chemokine receptor CXCR4 during cardiogenesis, and used FACS for the combined expression of CXCR4 and Flk-1 to select for cells of a cardiopoietic lineage from EB cells. The advantage of their approach is that the Bry-GFP cell line is not required. We are awaiting the approval of an MTA to obtain this cell line, but if we are unable to do so, we plan to use the alternate approach of sorting for CXCR4+/Flk-1+ cells to enrich for cardiac progenitors from EB cells. We will infect both unsorted and sorted EB cells with our GATA4 and Nkx2-5 vectors, and predict that we will get high transgene expression and enhanced induction of cardiogenesis. We will use RT-PCR to examine cells for the expression of cardiac-specific genes such as α-MHC, myocardin, and MEF2C. Observation of spontaneously beating cells will also be used to confirm the enrichment of cardiomyocytes.
Upon optimization of the differentiation of mES cells into cardiomyocytes, including MOI and timing of infection with GATA4 and Nkx2-5 vectors, and optimization of sorting conditions, we plan to collaborate with another laboratory to evaluate the ability of these cells to repair myocardial infarction in mice. Ultimately we wish to compare their ability to survive and form large grafts to undifferentiated mES cells or non-cardiac adult stem cells like MDSCs following intramyocardial transplantation, and predict that these mES cell- derived cardiomyocytes will contribute better to heart contractility and function.
Optimization of Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes for Use in Repair of Myocardial Infarction
While embryonic stem cells possess the ability to become virtually any cell type, many of the specific events involved in differentiation are unknown. The ability to direct the differentiation of stem cells into specific lineages would provide scientists with a powerful tool to generate cells for the treatment of many diseases and injuries. Stem cell-based treatment for myocardial infarction is particularly attractive due to the irreversible nature of the injury and the limited inherent regenerative capability of the heart. However transplant of undifferentiated stem cells is risky due to the possibility of teratoma formation, and the ability of these cells to engraft and participate in heart function is unknown. Researchers are beginning to understand the vital steps in cardiomyogenesis, and some of the earliest surface receptors and transcription factors expressed in cardiomyocyte progenitors have recently been identified. We hypothesize that by sorting murine embryonic stem (mES) cells differentiated via embryoid body (EB) formation based on expression of markers specific to the cardiac lineage, and ectopically expressing known cardiac transcription factors in these sorted cells using HSV vectors, we can produce a population of cells highly enriched for cardiomyocytes.
GATA4 and Nkx2.5 are two of the earliest transcription factors expressed in cardiac progenitors. They act together to regulate expression of cardiac specific genes in the developing heart, and exogenous expression of these two factors in mesenchymal stem cells has been shown to drive them towards a cardiac fate. We plan to construct HSV-1 vectors to express these genes in mES and embryoid body cells. HSV vectors are well suited for this work because they provide a pulse of gene expression which mimics the transient expression of developmentally regulated genes. The replication defective HSV vector JDββ has been shown to be capable of transgene expression in these cell types, and has a very low toxicity due to deletion of the immediate early genes ICP4 and ICP22, and reduced expression of ICP0 and ICP27, which are under control of an early (β) promoter. Previous work has also shown that this vector does not interfere with self-renewal or EB formation of mES cells, and does not alter expression of the mesoderm marker Brachyury (Bry). The latter is particularly important for our current work as cardiomyocytes are derived from this Bry positive mesoderm population.
Researchers have engineered a mES cell line which expresses EGFP under the control of the Bry promoter. They used FACS to sort cells based on expression of Bry along with Flk-1 to enrich for cardiac progenitors. A separate group used bioinformatic dissection to identify overexpression of the chemokine receptor CXCR4 during cardiogenesis, and used FACS for the combined expression of CXCR4 and Flk-1 to select for cells of a cardiopoietic lineage from EB cells. The advantage of their approach is that the Bry-GFP cell line is not required. We are awaiting the approval of an MTA to obtain this cell line, but if we are unable to do so, we plan to use the alternate approach of sorting for CXCR4+/Flk-1+ cells to enrich for cardiac progenitors from EB cells. We will infect both unsorted and sorted EB cells with our GATA4 and Nkx2-5 vectors, and predict that we will get high transgene expression and enhanced induction of cardiogenesis. We will use RT-PCR to examine cells for the expression of cardiac-specific genes such as α-MHC, myocardin, and MEF2C. Observation of spontaneously beating cells will also be used to confirm the enrichment of cardiomyocytes.
Upon optimization of the differentiation of mES cells into cardiomyocytes, including MOI and timing of infection with GATA4 and Nkx2-5 vectors, and optimization of sorting conditions, we plan to collaborate with another laboratory to evaluate the ability of these cells to repair myocardial infarction in mice. Ultimately we wish to compare their ability to survive and form large grafts to undifferentiated mES cells or non-cardiac adult stem cells like MDSCs following intramyocardial transplantation, and predict that these mES cell- derived cardiomyocytes will contribute better to heart contractility and function.
fun with procrastination
i haven't done any blogging this week so far because i just haven't felt like it. on sunday we did go to the pumpkin patch at schramm's, but it just wasn't a good time to go for a number of reasons, one of them being that it was crazy hot and sunny and just didn't set the right mood for autumn pumpkin picking. i thought it would be cute to put erik in orange for the occasion, but he got rather lost among the pumpkins.
he enjoyed testing out the pumpkins by sitting on them.
and he especially liked looking at the (stinky) little piggies.
we went into the market too, but didn't buy anything. not even a little pumpkin.
the rest of the week has been stressful/unpleasant for the most part. i think i had my first ever full blown panic attack on monday at the thought of my comps. i wrote up my abstract on tuesday, which is due next week. i think it's ok. i'm not crazy about it, but it's a start. i have about 3 weeks to complete my written proposal, and i'm pissing it away like i usually do. i can never do anything without a healthy dose of procrastination. i had my midterm in my stem cells class yesterday, so i spent a good bit of time procrastinating/worrying about/studying for that. when we got to class, we found out it was going to be a take home exam and didn't have to turn it in until the morning. it turns out i could have probably done really well if i hadn't used my notes. i can't imagine that i didn't get an A on it, although i know i missed one question for sure. this afternoon i did manage to collect all of the papers that i have so far that i will need to write my proposal. i am sure there will be many more, but for now my stack is only about 6 inches high, and i have read almost all of them. i am sure i will be reading them all again very soon! so here i am procrastinating again. it seems to be a theme in my life. i can't help myself. i probably have ADD or ADHD or some kind of other crazy thing. who knows?
he enjoyed testing out the pumpkins by sitting on them.
and he especially liked looking at the (stinky) little piggies.
we went into the market too, but didn't buy anything. not even a little pumpkin.
the rest of the week has been stressful/unpleasant for the most part. i think i had my first ever full blown panic attack on monday at the thought of my comps. i wrote up my abstract on tuesday, which is due next week. i think it's ok. i'm not crazy about it, but it's a start. i have about 3 weeks to complete my written proposal, and i'm pissing it away like i usually do. i can never do anything without a healthy dose of procrastination. i had my midterm in my stem cells class yesterday, so i spent a good bit of time procrastinating/worrying about/studying for that. when we got to class, we found out it was going to be a take home exam and didn't have to turn it in until the morning. it turns out i could have probably done really well if i hadn't used my notes. i can't imagine that i didn't get an A on it, although i know i missed one question for sure. this afternoon i did manage to collect all of the papers that i have so far that i will need to write my proposal. i am sure there will be many more, but for now my stack is only about 6 inches high, and i have read almost all of them. i am sure i will be reading them all again very soon! so here i am procrastinating again. it seems to be a theme in my life. i can't help myself. i probably have ADD or ADHD or some kind of other crazy thing. who knows?
Saturday, October 11, 2008
unseasonably warm
today was a very quiet saturday. i am enjoying the procrastination (in a guilty sort of way) of not having yet started working on my comps. it will be over on monday. anyway, it was an amazingly beautiful day for october. fall is my favorite time of year, and days like these are exactly why. the nights are cool and perfect for sleeping, and the days are warm and sunny. this week at least. overall erik and i had a good day today. i decided to keep it as low key for him as possible since he hasn't been feeling well and has been terribly ornery all week. we did some grocery shopping this morning and i even got the shopping cart that looks like a car because he enjoys it (even though i spent the entire time bumping into people or running over their toes since i can't steer the stupid thing). nonetheless he had fun turning the little steering wheel and making quiet vvrrrooom vvrrooooms around the store. after his nap we went out to schenley park to check out the "obama-thon" ... only kidding. i just wanted to take erik somewhere he could run around and play without worrying about him running into the street. we took a nice walk and ended up at probably the nicest and largest playground in the park and he had lots of fun running amok.
and we went through a little tunnel ...
with a gnome in it.
i made meatloaf, green beans, and baked potatoes for dinner, which all three of us enjoyed when peter got home from work. after the dishes were all cleaned up, we took advantage of the beautiful warm evening and went to the rita's in oakland, perhaps our last visit there this year. erik really loved his dessert ... he shared my peach ice and peter's twist custard. yummy! not a bad day at all. maybe tomorrow we will go to the pumpkin patch!
and we went through a little tunnel ...
with a gnome in it.
i made meatloaf, green beans, and baked potatoes for dinner, which all three of us enjoyed when peter got home from work. after the dishes were all cleaned up, we took advantage of the beautiful warm evening and went to the rita's in oakland, perhaps our last visit there this year. erik really loved his dessert ... he shared my peach ice and peter's twist custard. yummy! not a bad day at all. maybe tomorrow we will go to the pumpkin patch!
Thursday, October 9, 2008
ARGH
not a good week. more erik throwing up. got notification of my comps. i'm feeling very cranky. here are some pretty flowers.
Wednesday, October 8, 2008
apple and arts
on sunday, peter, erik, and i headed out to delmont for the annual apple and arts festival. this was our 3rd year going together and it's always a great time. it started out particularly good when we met up with my mom and she asked peter if he would judge the baking contest (apple themed, of course!). i immediately asked if i could do it, and luckily peter was happy to let me. it was like a dream come true! here's me and my fellow judges:
we got to taste 13 delicious desserts and it was a real challenge to pick a winner. kudos to all who entered the contest - you all did a terrific job!
immediately after that, peter, erik, and my mom were hungry and ready to eat, so i took one for the team and partook of some more delicious victuals, namely a fried dough salami sandwich. YUM! peter, who wisely saved room, also had some great chicken on a stick and some of my mom's beef. unfortunately we didn't get our apple dumplings in time and missed out. luckily we learned from previous years and got our cider early in the day so as to avoid the long lines at the end of the festival.
but enough about the food! while judging the baking contest was awesome, the highlight of my day was taking erik to the petting zoo and seeing how excited he got to see and pet the sheep, goats, geese, donkeys, ducks, chickens, and cute furry scottish cow thing.
erik's granny was sweet and suggested that he should go on the pony ride, and doubly sweet in missing it the first time because she was busy being the boss lady so he had to go again. he LOVED it! i thought he would be either bored or (more likely) freaked out by it, but he grinned from ear to ear and cried when it was time to get off. i am so excited that maybe in a few years i will have someone to go horseback riding with!
towards the end of the day, chrissy, morgan, marisa, and maggie came and we got to spend a little time walking around with them. erik hadn't napped and was cranky and tired, but still enjoyed seeing his cousins.
it was a long day, but the weather was perfect (third year in a row!) and we had a great time as always! thank you to my mom for making it all possible! without her, apple and arts would just be ple and rts ... or something like that :)
we got to taste 13 delicious desserts and it was a real challenge to pick a winner. kudos to all who entered the contest - you all did a terrific job!
immediately after that, peter, erik, and my mom were hungry and ready to eat, so i took one for the team and partook of some more delicious victuals, namely a fried dough salami sandwich. YUM! peter, who wisely saved room, also had some great chicken on a stick and some of my mom's beef. unfortunately we didn't get our apple dumplings in time and missed out. luckily we learned from previous years and got our cider early in the day so as to avoid the long lines at the end of the festival.
but enough about the food! while judging the baking contest was awesome, the highlight of my day was taking erik to the petting zoo and seeing how excited he got to see and pet the sheep, goats, geese, donkeys, ducks, chickens, and cute furry scottish cow thing.
erik's granny was sweet and suggested that he should go on the pony ride, and doubly sweet in missing it the first time because she was busy being the boss lady so he had to go again. he LOVED it! i thought he would be either bored or (more likely) freaked out by it, but he grinned from ear to ear and cried when it was time to get off. i am so excited that maybe in a few years i will have someone to go horseback riding with!
towards the end of the day, chrissy, morgan, marisa, and maggie came and we got to spend a little time walking around with them. erik hadn't napped and was cranky and tired, but still enjoyed seeing his cousins.
it was a long day, but the weather was perfect (third year in a row!) and we had a great time as always! thank you to my mom for making it all possible! without her, apple and arts would just be ple and rts ... or something like that :)
Monday, October 6, 2008
pittsburgh's 250th birthday
pittsburgh really knows how to celebrate a birthday - by blowing stuff up! on saturday night, pittsburgh celebrated it's 250th birthday with perhaps the largest fireworks display in US history. fireworks were set off from 17 different locations including the tops of skyscrapers, barges in all three rivers, and bridges. i had planned on watching the display from the building where my lab is, right along the monongahela next to the hot metal bridge, however by 10pm when they were set to start, i was exhausted and decided to stay home and watch them with peter from the upstairs at our house. even from 3 miles away and through a tree, they were amazing to watch. they lasted about 30 minutes, and were synched to music which we unfortunately did not hear. it was really weird to see the flashes of light and not hear the "booms" until significantly later. we really enjoyed the show! i took a few pictures with my "little" camera. they're not good by any stretch of the imagination, but they give you an idea of our perspective:
this last one shows the fireworks being launched from the tops of several buildings:
this last one shows the fireworks being launched from the tops of several buildings:
happy birthday pittsburgh! - part 2
on saturday afternoon, after erik's nap, we went down to the waterfront to walk around the court, across the footbridge, and a little ways down the trail past the homestead grays bridge.
erik enjoyed playing in the gravel on the path and watching the fountain, and i was happy to get to check out some of the sculptures by romero britto that were on exhibit around the center of the courtyard (while making sure erik did not jump into the fountain). romero britto is a brazilian artist whose works are temporarily on display at the waterfront in homestead. i don't know much about him, but found his brightly colored, neo-pop sculptures very pleasing to look at. this one was my favorite:
here are a couple others:
here are some pictures i took of erik:
erik enjoyed playing in the gravel on the path and watching the fountain, and i was happy to get to check out some of the sculptures by romero britto that were on exhibit around the center of the courtyard (while making sure erik did not jump into the fountain). romero britto is a brazilian artist whose works are temporarily on display at the waterfront in homestead. i don't know much about him, but found his brightly colored, neo-pop sculptures very pleasing to look at. this one was my favorite:
here are a couple others:
here are some pictures i took of erik:
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