Protoreaster

Thanks for the visiting the thread sen5241b. So far I can say that the data suggests that Bone Morphogenetic Protein plays a role within the proliferation of starfish cells. We are currently looking at ways to collect causal evidence for Bone Morphogenetic Protein’s ability to increase the rates of starfish cell regeneration and cell differentiation. Our results point towards a promising conclusion so what lies ahead will be interesting! My thread on the education forum goes into more detail on the specifics of our future plans that will allow us to conclude whether BMP is truly affecting regeneration rather than proliferation.

Hi Mogurnda, thanks for taking a look at our project. Interestingly, my partner and I were initially looking at using Noggin to inhibit BMP signaling in regenerating cells to determine whether there would be a difference in regeneration rates of starfish cells treated with Noggin when compared to the rates of those growing without Noggin. We decided to use BMP instead with the hopes of investigating its potential use in a biomedical setting for tissue therapy and because our Singaporean research partners were using chemical compounds to increase rates of earthworm tissue regeneration (We need to engineer our experimental designs to be comparable after collecting data and results). Using Noggin next school year and possibly during the summer sounds like a great course of action to determine BMP’s definitive role in the process of regeneration. We are also thinking about looking at the proteins and genes further downstream in the BMP pathway that are activated when BMP binds to receptors on cells to collect causal evidence for BMP leading to cell differentiation. We are currently doing background research on the SMADs in the BMP pathway and will get back to you with a more definitive answer for our future endeavors. I subscribed to your solar sea slug thread, you and your team look like you’re having a great time while working. Thanks for your experienced insight!

Origami, I should have taken some better pictures of the overflow system in my original post! The PVC elbow in the specimen tank is actually the backup if the current water outlet is blocked by too many starfish crawling over the opening. The current water outlet has a strainer which functions to keep starfish from blocking water from freely flowing into the sump. We only used two outlets because we ran out of PVC pipe left over from my father's projects. If the starfish were to group around the outlet, the PVC elbow serves as the backup. Here are better pictures of the system, including one of the starfish crawling over the strainer: I guess if the starfish decided to block off the current strainer and PVC elbow we would have a problem... with the current number of stars in the tank that doesn't seem to be physically possible but I wouldn't put anything past these guys. I think I'll end up adding another strainer to the overflow especially if we get more sea stars. We transfer the stars to the lab in a cooler filled with tank water from the home system. The lab system has a salinity matched to that of the system at home, but variations are most probably present due to the differing tank dynamic between the two locations (number of sea stars/water capacity). When culturing our cells initially we were unaware of the sensitivity the sea stars would exhibit towards water changes and began cutting them the day we brought the stars to the lab environment. Our lack of awareness could have contributed to our lacking success with cell cultures in January/February. We found that any sort of water change within a week of cutting starfish for tissue samples condemned the sea stars to an almost certain death due to minor changes in salinity, so we learned to be careful about timing feeding sessions of the injured starfish. Our newer method for cell culture involved allowing the starfish to acclimate to the lab’s tank environment for a period of 2-4 days before cutting and taking tissue samples. We however did not use the standard procedure of incremental water addition (from the lab tank environment) to our livestock “carrier” to initially acclimate our stars to the lab tank environment. We had not considered the transfer process as a potential for sea star injury. It would be interesting to determine whether or not this procedure, surely placing our livestock under a state of stress, has an effect on baseline levels of BMP concentration. Injured starfish tissue has a higher concentration of BMP than that of uninjured starfish tissue indicating that the starfish might be producing the BMP to repair the injured tissues. When examining possibilities for bias in our data due to the transfer of starfish having a potential effect on baseline levels of BMP concentration, we believe that because all of the starfish cells in one trial come from the same organism at the same time, the initial amounts of BMP in the injured tissues would appear consistent (if the process of transfer is actually affecting these base levels of BMP concentration). We performed a Mann Whitney statistical test to determine whether the median values for percent surface area of cell coverage was statistically different across our first and second trials. We expected the percentages of cell coverage to change as the cells were allowed to grow in media with different concentrations of BMP but we really want the two trials to procure similar percentages of cell coverage for each BMP concentration on each day we took pictures of our cells. The Mann Whitney test yields a “P Value” which is indicative of statistical significance for a comparison of the medians of two populations. Hypothetically, a P Value below 0.05 is indicative of statistical significance and in reference to our project would mean our trials had produced inconsistent data with varying initial conditions for our cell cultures and variation in our experimental design. However, our yielded P values are all above the value of 0.05 suggesting that the the percentage of cell coverage we observed in first trial did not vary significantly with the percentage area of cell coverage seen in the second trial. We believe that if a bias in our data was present, it most likely would have been present in the comparison of consistency across the various trials we conducted to compare growth rates of cells in varying concentrations of BMP.

Origami, thanks for the quick response. The work my partner and I want to complete over the summer includes collecting data that serves as causal evidence for our BMP concentrations affecting cell differentiation. Instead of simply looking at increases in surface area of cell coverage for each BMP concentration over the course of 10 days (which is indicative of proliferation), we will count the numbers of differentiating stem cells and compare these counts across concentrations of our BMP application. Currently our data is a bit ambiguous, as the proliferation that appears to be caused by BMP could easily be a form of tumor-like growth, however the absence of cells growing on top of each other serves as a good indicator that future data could suggest that BMP indeeds plays a definitive role in cell differentiation. We haven’t given much thought to finding a procedure that measures uptake/metabolization of BMP overtime. We would have to find the intracellular molecules and/or mRNA that would be produced as BMP attaches to receptors on cells, then we might be able to use a Western Blot to detect proteins that are upregulated by BMP metabolization. Our background research indicates that the starfish is unusual in its ability to produce BMP within injured adult tissues and it is rare to find this protein naturally produced in injured adult tissues of other animals. We would love to continue our research after the school year is over (6/14/16 is our last day!) but we will likely run into a problem with funding as the AOS research grant is only “refreshed” near the end of the summer. I had a great time learning more detailed procedures for fish husbandry. However, my partner and I agree that presenting our research at the AOS Symposium/Science Fair was the highlight of our year. We had the most fun conducting our presentation as it includes talking with other people who are just as interested in research as we are.

Hello all, I am a junior at Dominion High School and currently attend the Loudoun Academy of Science (AOS), a magnet science and math program. One of the keystone aspects of the AOS is the required two year research projects each student must investigate as a junior and senior. These projects can cover a variety of subjects ranging from environmental science to computer programming and math modeling. I was fortunate enough to be included in a partnership program for my research project. The program allows two AOS students to collaborate with two Singaporean students on comparative research projects. My partner and I are nearing the end of our first year researching the effects of a protein on the rates of starfish cell regeneration, and by the time August rolls around we will travel to Singapore to present our research as a team of four to Singaporean science judges. In May we submitted our project to the Google Science Fair (Link to our Google submission here: https://goo.gl/wCI5jA. In early June we participated at the AOS science symposium and were awarded first place in the category of Animal Science. We hope to apply to the Siemens science competition in October once we gather more data. The AOS has had some winners of Siemens and other high profile competitions (Intel's International Science and Engineering Fair) so we are crossing our fingers! I posted on the education forum to bring to light some of the comprehensive work currently conducted in the neighborhood by students with an interest in marine biology and fish keeping. By conducting our project, my partner and I solidified personal enthusiasm to pursue fish keeping and to further the efforts of other student researchers with the same passion that we’ve discovered. There are quite a few other students of Loudoun County and the Loudoun Academy of Science (AOS) who have completed projects that are relevant to the husbandry of aquatic creatures and keeping our oceans clean so I would love to encourage them to post/join here about their journeys, or even ask for advice on successful animal husbandry (if the WAMAS community would be interested). After realizing how much our backgrounds in fish keeping aided the progress of our project, It would be a dream for my partner and me to facilitate some sort of connection between the AOS and WAMAS community for researchers with an enthusiasm for marine biology For those of you who check out my post on the education forum, thanks for listening to my story! I look forward to joining the community and formally entering the hobby! Here is a link to my thread in the Education forum: http://wamas.org/forums/topic/78553-loudoun-academy-of-science-starfish-husbandryregeneration-investigation/ Best wishes, Thanks for listening to my story, Dale “Protoreaster”

Tl;dr: Current junior at the Loudoun Academy of Science conducting a research partnership that will travel to Singapore to present research. Currently investigating the effects of a unique protein on the rates of starfish cell regeneration, identified one of the proteins behind starfish regeneration and established its potential for regenerative tissue therapy, hoping to facilitate a connection between WAMAS and Loudoun County student researchers with a passion for marine biology. I posted an introduction on the "General Discussion" forum if any were interested. Tank Specifications My partner and I began to work together on a project investigating starfish regeneration in September/August of 2015. Before we could begin to work we had to establish an efficient means of starfish husbandry at one of our houses (our school does not have extensive room for large fish tanks). I come from a background that was rooted in fish keeping which allowed our partnership to progress towards the “wet lab” stage of our investigation pretty quickly. My father (Traveller7) used to frequent these forums and ReefCentral and was known for his care of anemones & clownfish. His prior hobby leftovers allowed my partner and me to use old tanks and pumps to immediately begin care for our starfish. The progress of our project would have most definitely been hindered had neither my partner nor I had some sort of background in Marine Biology. For our tank set up at my home, we set up a two tank system to ensure water quality maintenance would be less of a problem as starfish were introduced to our system. I modified a vertical tank stand that a previous AOS student used to investigate sexual hermaphroditism in clownfish. My father and I placed a 48x13x13 custom made Glass Cages - Euro Brace tank on top of the modified tank stand. The main tank was plumbed to the lower sump tank through outlets originally intended for a laminar flow model of water circulation. These outlets are connected with PVC pipes allowing water to flow down into the tank placed inside the stand, a 40 gallon breeder used as a sump. These PVC pipes have nylon socks attached to the ends sitting in the sump tank in hopes of capturing detritus from starfish feed debris. A basket of porous ceramic balls sits under/next to the water inflow to allow bacteria nitrifying bacteria to proliferate. A used RLSS6-I skimmer sits in the sump to help maintain water quality in the system. The water is heated at a stable temperature to model the temperature of the environment from which the starfish (Chocolate chip Sea stars/Protoreaster nodosus) come. The water in the sump is pumped back up to the top tank holding all livestock with a 600 gallon/hour pump. The Glass Cages tank sitting on top of the stand held from 5-11 starfish at any one time (this number would vary based on the time of year, decreasing as we would bring them into our school to begin work). All the goons are lining up for their picture here: We procured our starfish from Blue Ribbon Koi located near Aldie, Virginia (Shoutout to John for taking such good care of us!). The starfish were initially fed every week with frozen shrimp gels that John provided but we later switched to algae tablets as the starfish would have problems eating the frozen shrimp. As we began to start “wet lab” (actual manipulation of starfish for our investigation) my partner and I brought the starfish to school and placed them in a used CPR 6 / Nano Tank. My partner and I placed a pump inside the tank to facilitate oxygenation and used a nylon gauze and half moon sponge as a mechanical filter for starfish food/innard detritus. This tank held from 1-3 starfish at any one time and was used to care for starfish as they would be cut when we collected coelomic epithelial starfish cells from them. For future work on the tank at my home, I'm thinking about installing an auto-top off into the tank system, which can be filled with the water purification system that my father had installed for his projects with clownfish/anemone care. Any comments with regards to tank care would be very helpful! Research Specifics In early September (2015) , my partner and I initially heard that we would be holding a joint investigation with our Singaporean partners who were looking at properties of earthworm regeneration. They intended to experiment on the macroscopic organism itself so we also planned to conduct an in vivo (within the living) investigation involving manipulation of the starfish itself. The Singaporeans travelled to our school and arrived in early November to plan the course of our parallel investigations to ensure that we would be able to successfully compare our results the next summer in August. However, as a partnership we decided it would add complexity to the research comparison by comparing an in vivo, macroscopic perspective of earthworm regeneration (Singaporean partners) to an in vitro, microscopic perspective of starfish cell regeneration (American partners). By mid November our Singaporean partners returned home to attend school and begin their side of the research investigation. My American partner and I would continue planning our research until January (2016) when we would actually begin the “wet lab” portion of our investigation. My American partner and I are looking at the effect of this molecule called Bone Morphogenetic Protein (BMP) on the rates of regeneration of injured starfish coelomic epithelial cells. This protein, at first, appears to have no significance but a few things need to be noted. BMP is a molecule that is present in almost every living organism and appears to be “evolutionarily conserved” across the development of life seen in your typical tree of evolution. This protein is vital to the embryonic stage of development, as it “tells” undifferentiated, flexible embryonic stem cells to become every kind of cell necessary for the organism to flourish after developing as an embryo. It will tell these cells to become nerve cells, muscle cells, and every other kind of cell we have within our bodies as adults while organizing the position of these cells in the embryo. What’s even more interesting is how the starfish will express this protein within injured tissues even as an adult while in almost all other organisms it's rarely present after the stage of embryonic development. After understanding the role of this protein in embryonic cell differentiation (stem cells becoming other kinds of cells) we wondered if this protein allowed adult starfish stem cells to differentiate and regenerate in a way that modeled embryonic development. That got my partner and I thinking: “Could it be the presence of this protein within injured adult starfish tissues that allows the starfish to uphold its reputation as a creature with incredible regenerative capabilities?”. We wanted to determine whether BMP was one of the main proteins behind the natural “miracle” of starfish regeneration. While also determining its purpose within injured starfish cells, we wanted to determine whether this protein had the opportunity to be applied as a medication to injured tissues, potentially those of injured people. To embark on this investigation we had to formulate a method of starfish cell culture. In the process of cell culture we injure the starfish cells, removing an additional procedure for injuring cells after initial cell culture. Our first attempt to culture starfish cells in January turned out poorly. We encountered a variety of problems with starfish cell culture which stemmed from the fact that keeping adult echinoderm cells alive remains a relatively unstudied field. We initially were confused as to why that was the case, but quickly found out that without a background in fish keeping or the will to put in effort to establish an effective means of echinoderm husbandry researchers would run out of livestock to experiment upon (or the patience to repeatedly fail and change small aspects of cell culture to try and get experimental procedure to work correctly). Here’s a picture of me in the sterilized cell hood during our first attempts at cell culture: Here’s another picture of the sterilized hoods used for cell work/research: Cells survive in small plastic flasks or disc-shaped plates that are filled with their “food” called cell media. Here’s a picture of our cells in a 24 well-plate under the microscope we used to take pictures of our cells. One of the most vital parts of our investigation had to be the creation/adaptation of adult starfish cell media. We adapted a composition of “cell food” from what has previously been used to keep embryonic echinoderm cell cultures alive in the lab, sea urchin embryos to be precise. By using a modified cell media we found that we could culture adult starfish cells in the lab and watch them regenerate after being taken from starfish flesh. Here is a picture of the laminar flow bacteriology hood we used to manipulate and cut the starfish during the process of collecting cells. The method with which we collected our starfish cells also provided us with a roadblock. Here’s a link to a video of my partner collecting starfish cells: https://www.youtube.com/watch?v=knGNbQWxBHM&feature=youtu.be Many projects that look into cellular biology are conducted with cells purchased from established organizations but as explained previously, starfish cell cultures are not yet sold due to the finicky nature of culturing adult echinoderm cell lines. We initially used a mechanical method for cell separation, pulverizing fragments of the starfish coelomic epithelium (the tissue lining the inside of the starfish arm cavity) we removed from amputated starfish limbs then separating the cells from debris by using a cell strainer. This method proved ineffective from January-February, leading us to use a chemical method for cell separation. We decided to use a chemical called collagenase to dissolve the material holding cells to the fragments of epithelia we pull from the starfish arm. It was March at this point and we had missed the opportunity to apply within the AOS for the Regional Science Fair as we spent an extended period of time trying to get our method of cell culture to work out. Although we hadn’t collected data by the time some other juniors had, we learned about the aspects of failure and success in experimental design. Here’s a picture of what our cells look like (the shapes that are circular and black/blue are differentiating starfish coelomic epithelial cells). By the end of March we began to test the effects of BMP on our injured starfish cell cultures. We added dehydrated BMP to our cell media in different concentrations to test our hypothesis that different concentrations of BMP applications to injured starfish cells would increase rates of starfish cell regeneration. Every two days we take pictures of cells that grow in cell media that has varying concentrations of our BMP application. After analyzing the images for changes in percent area of cell coverage over the course of ten days (for a total of 3 trials) we determine (statistically) that starfish cells growing in increasing concentrations of BMP show increasing trends of statistical difference from starfish cells growing in media without an application of BMP. The difference in growth rates serves to indicate that BMP indeed has an effect on cell proliferation of adult starfish cells after injury. Furthermore, such a difference suggests that the normal expression of BMP in regenerating adult starfish cells could exist to facilitate the regeneration of tissue, which could be augmented with additional application of BMP to injured cells. Lots of future work lies ahead of my partner and me. While we have evidence that BMP affects cell proliferation, we want to be certain that it is affecting the differentiation of stem cells rather than an alternative element of cell proliferation like unwanted tumor growth. While we observed differences in stem cells counts in our images of the regenerating cells, we want to run more trials and run statistical tests on more data to come to a more complex conclusion of BMP’s effects on injured starfish cells and procure more causal evidence of BMP’s function within these cells. We hope to conduct this further research this Summer but the AOS research funds have run out until the fall: Our investigation’s near future remains a mystery unless we find a solution. I remember my principal, Mr. Wolfe (The AOS research guru), emphasizes the need to find a reason for somebody to, “pay you one million dollars to conduct your research”. Our project has relevance in the field of regenerative medicine by finding an alternative perspective towards adult tissue therapy. After collecting causal evidence in additional trials, we want to begin applying our research to adult human cells. We want to determine if this protein or a combination of this protein and other chemicals can accelerate the rates of injured human tissue regeneration. I described earlier how adult echinoderm cell cultures are not sold and investigations requiring them forces researchers to resort to their own methods of echinoderm cell culture. Adult echinoderms are known for their regenerative properties, making them a prospect to facilitate the uncovering of secrets to adult cell regeneration. My partner and I are also currently looking at a way to use cryopreservation to freeze our starfish coelomic epithelial cells and determine whether they are viable for research after freezing. If we are able to use our modified cell media to grow the cells even after freezing, we open up the doors to an untouched perspective of cell regeneration in the field of regenerative medicine. Essentially, widespread transport, sale, and research of these cells and other adult echinoderm cell lines could be possible by using our adapted method of starfish cell culture. My partner and I are currently looking at the viability of these cells after cryopreservation within our last week of school so we are hopeful that we can gather some data within our tight time frame! Thanks for listening to my story, Dale “Protoreaster”