Science Buddies: "Ask an Expert"

Dear ScienceBuddies,
I am a student in Grade 8 and I chose the project Creating a Kidney: Using Stem Cells to bioengineer a vital organ for my science fair this year. I made a modification and so instead of using the information about the kidney, I wanted to bioengineer a brain, or at least parts of it. I did this because I wanted to tie my project with diseases like Parkinson's and Alzheimer's but I am having a few problems.

First of all, I cannot find anywhere the supplemented soluble factors needed when creating Embryonic stem cells into dopaminergic neurons or nerve cells that produce acetylcholine. Can you help me with that as there is no research paper I can find to help me with that information.

I also cannot seem to find the alpha and beta integrin subunits for a brain or certain parts of it and therefore I cannot find the ECM proteins that they bind. This means I cannot use Amazonia without information, and I cannot use any of the 4 tables given because of these problems. Can you help me in any way?

also because there is no experiment associated with this project is it fine to do this type of science fair project in an actual regional science fair.

Radhika

hi can anybody help me with the project i am doing please?? It will be great help.

It's going to be very difficult for you to create an actual brain. Differentiation of ES cells is a highly involved process and very cost prohibitive. Even use of a retinoic acid for the nondirection differentiation of ES cells doesn't guarantee 100% conversion into neural progenitor cells. Nevertheless, could you please provide the current protocol you're using to differentiate your ES cells? Are you using mouse cells? There are a lot of scientific papers (And I mean A LOT of papers) that study the differentiation of ES cells into neural precurors cells. I think if you're very determined to get this done, it's certainly doable.

The follow statement also needs much more clarification before anyone can help you with it:
"I also cannot seem to find the alpha and beta integrin subunits for a brain or certain parts of it and therefore I cannot find the ECM proteins that they bind. This means I cannot use Amazonia without information, and I cannot use any of the 4 tables given because of these problems. Can you help me in any way?"

Could you please provide more details about your project? What is your question? What is your hypothesis?

Hi Radhika. You have undertaken quite a difficult project in trying to come up with a recipe for turning stem cells into neurons. Stem cells can become almost any other type of cell, but the proteins and other factors that you have to give them to do this can be quite different from say a kidney cell compared to a neuron.

You are really starting from the beginning on this project since you will need to do a search of the scientific literature just as the person who designed the project on the kidney had to do--only you have to find the soluble factors and extracellular matrix proteins that are needed to differentiate stem cells into neurons. This information is out there if you know how to use Google Scholar and PubMed. We can try to help you focus in on the best sources and explain things that you don't understand. Actually doing a project like this will teach you a lot more than just using information that has already been collected.

I did a quick search on converting stem cells to neurons and here are some of the potential sources I came up with:

1. Engineering Microenvironments to Control Stem Cell Fate and Function (http://www.stembook.org/node/484)
This article is pretty general and very technical, but I did find the following specific details within the article about how to convert eSCs to motor neurons: "Differentiation of motor neurons from primitive ectoderm requires progression through two intermediate stages, each guided by a distinct set of soluble factors. Ectodermal cells first acquire a rostral neural fate through signaling by BMP, FGF, and Wnt proteins (Munoz-Sanjuan and Brivanlou, 2002). The next transition is retinoic acid-mediated caudalization, followed by progression to terminally differentiated motor neurons in the presence of Shh (Briscoe and Ericson, 2001). In a landmark study, Jessell and colleagues were able to guide mouse ESCs down this pathway by applying these soluble factors in a step-wise manner that emulates natural neural development, resulting in the in vitro generation of motor neurons that can survive and engraft in vivo (Wichterle et al., 2002)."

The soluble factors used were
- BMP (bone morphogenetic protein, https://en.wikipedia.org/wiki/Bone_morp ... ic_protein
- FGF (fibroblast growth factor), https://en.wikipedia.org/wiki/Fibroblast_growth_factor
- Wnt ("The name Wnt was chosen because it is a combination, or portmanteau, of int and Wg and stands for Wingless-related integration site.[2]"), https://en.wikipedia.org/wiki/Wnt_signaling_pathway
- retinoic acid (RA, https://en.wikipedia.org/wiki/Retinoic_acid)
- Shh (sonic hedgehog protein--yes, that really is its name!), https://en.wikipedia.org/wiki/Sonic_hedgehog

2. Neural Stem Cell Response to Extracellular Matrix (http://gradworks.umi.com/35/14/3514762.html)
This is also a highly technical article but it is important because it lists some of the matrix factors that stem cells need to become neurons. The 'matrix' is just the tissue within an organ that a cell grows in, but it is important because the matrix (abbreviated ECM) produces protein factors that are necessary for the survival and conversion of stem cells to some other cell such as a neuron. Here's the relevant quote from the article:

"The third aim examines the importance of signaling from the laminin integrin α6β1, an integrin chosen due to the importance of laminin in the nervous system. The results identified qualitative and quantitative increases in the production of laminin, fibronectin, and type IV collagen due to the dynamic stimulation of endothelial cells (aim 1)."

Here are the ECM factors that were examined in this system and they are similar to those needed for the kidney system in the Scibuddies project:
- laminin integrin alpha6-beta1
- fibronectin
- type IV collagen

I hope this helps to get you started on the project. Try doing your own searches and see what you can come up with. I’m sure you will find variations on these stem cell recipes from one lab to another. Make a list of things you don’t understand and post it and we will try to explain them. Scientists have their own jargon and stem cell research is pretty complicated so you will learn a lot from this.

Good luck!

Sybee

Thank you so much Sybee for the informative response and your sources were really helpful for both information/knowledge and the project. I have just recently started this project and have started to formulate my hypothesis. This is what I have got so far:
Hypothesis #1 (Curing Parkinson’s): If the neural stem cells are grown in a laboratory and then transplanted into the basal ganglia of a diseased brain, then the stem cells can replace the other damaged neurons in the substantia nigra, restore motor functions in the body, and potentially cure Parkinson’s disease.
Hypothesis #2 (Curing Alzheimer’s): If neural stem cells are grown from an Embryonic Stem Cell or Induced Pluripotent Stem Cell and then transplanted in the major parts of the and the forebrain, then the stem cells can either heal the other damaged neurons or replace them throughout the brain, potentially curing Alzheimer’s disease.
Hypothesis #3 (Growing the Stem Cells): If the stem cells grow in a very sterile environment where the necessary soluble factors are constantly added and the surroundings of the stem cells are the exact same at all times (the temperature), then the necessary stem cell type will be derived from an Embryonic stem cell and can be used for any transplants.

Also for my variables and constants, I have got this:
Independent Variable: The concentration of the added soluble factors, The soluble factors added into the media to differentiate the cells, Medications and dietary requirements taken by the patient to prevent patient rejection
Dependent Variable: The type of stem cell differentiated from the Embryonic or Adult stem cell, The number of neural progenitors that grow on the petri dishes, The effectiveness of the new stem cells in the brain
Constants: The temperature at which the cells are all cultured at, The settings in the incubator, The health of the embryo or human from which the embryonic stem cells or the adult stem cells are taken from, The environment around the stem cells while they are growing (the sterility), The immune system of a human/embryo, Type of petri dish used to grow the cells

Can you pls reply back to tell me if these are ok and i will post back more if i need your help again.

Radhika

P.S. the Amazonia database had recently crashed or something happened to it I cannot access it anymore so can you pls check this problem

Hi Radhika,

You have proposed an ambitious, professional and thorough stem cell project for treating human neuronal diseases, but I would like to make one comment based on my own experience. When I have written research grant proposals and submitted them to the National Institutes of Health for funding, the most common criticism I have gotten is that the project lacks focus and is attempting too much. So now I have learned to stick with a single hypothesis and to test it by three or four separate aims.

I think you should choose just one disease to tackle at the start, say Parkinson’s, and to utilize a single type of stem cell, preferably iPSCs because that avoids the ethical problem associated with using human embryos and allows you to transplant the patient’s own cells without risk of rejection. Here is some information on it: https://www.michaeljfox.org/understandi ... stem-cells

As you are probably aware since you have been doing a lot of reading about stem cells, there are risks in using iPSCs and as far as I know only one clinical trial has been approved—a study in Japan testing iPSCs for treatment of the eye disease, macular degeneration in September 2014 (http://www.nature.com/news/next-generat ... al-1.15897), so the initial results should be known later this year.

Transplanting neuronal cells derived from iPSCs into the brain carries a real risk of causing tumors since these iPSCs are able to grow and divide without limit, at least in a Petri dish. This seems to be the main concern of scientists so I would be sure to work on ways to avoid that in your proposal. Do a PubMed (http://www.ncbi.nlm.nih.gov/pubmed) search and try to find a recent review on iPSCs that includes information about how to prevent tumor formation. It will make your proposal much stronger if you can discover the right mix of factors to grow new neurons for Parkinson’s patients without giving them brain cancer.

I have worked with mesenchymal stem cells (https://en.wikipedia.org/wiki/Mesenchymal_stem_cell) from bone marrow but have not grown iPSCs so do not have firsthand experience there. The references I sent you before seem to contain reasonable growth and differentiation factor recipes, but I strongly suggest that you read as many other good articles as you can to find out what variations have been tried and what the results were.

When you get stuck trying to understand the science jargon and confusing terminology used by many writers, just make a list of your questions and post them and we will try to make sense out of it. This is a really exciting research area and it will be great if you succeed in creating a scientifically feasible proposal. Plus it is good practice for writing a grant proposal later on if you do decide to become a biomedical researcher.

I tried reaching the Amazonia site at: amazonia.transcriptome.eu/search.php
and it timed out so there must be some problem with the software. I would keep trying it. In the meantime, here are some other searchable genetic database sources you can try:
http://www.ncbi.nlm.nih.gov/guide/genetics-medicine/
http://www.ncbi.nlm.nih.gov/guide/genes-expression/
http://www.ncbi.nlm.nih.gov/gene/
http://www.nextprot.org/

Good luck!

Sybee

Hi Sybee thx for the information just one quick question I had. If in Amazonia there are 2 spots in the Embryonic section should I use the top or the bottom. Thanks for all the useful sites they really were good studies to read.

So, Amazonia is finally working again. Let's hope it stays up.

I'm not sure how to answer your question because I don't know exactly what you are looking at. Can you post the search terms you used to get to the particular page that you are referring to? I don't use Amazonia so I may not be able to help you unless I can see exactly what you are looking at. You could attach a screen shot and maybe i could figure it out. Explain what you did in detail on Amazonia and maybe someone else who is experienced with this software can answer your question.

Sybee