The Effects of Ascorbic Acid, Alpha-Tocopherol, and Ergocalciferol on
the Stem Cell Growth of Dugesia Tigrina
For Cardiovascular Disease (CVD) Research
Scienteer Student ID: 150129
Question and Hypothesis
Background
The application of the concept of stem cells to the field of regenerative medicine is key to a future where
cell systems can be regrown for certain medical scenarios. The study of stem cells is an extremely complex
science that researchers are still learning about each and every day. Flatworms can be manipulated for the study
of such a complex phenomenon. This project is primarily centralized around the role stem cells play in tissue
regeneration (wound healing), and learning how to enhance this natural ability via. antioxidant substances.
Stem Cells, Classification, and Roles
Stem cells are essentially cells that do not have a specific role, cells at their earliest stage. These cells
then differentiate, and according to genetics, create cells with specific functions. For example, in the human
kidney, there are about twenty-six different types of cells with different roles. These cells then make up tissues,
which then make up organs. Basic biology.
Stem cells are typically categorized in three primary ways: totipotent, pluripotent, and multipotent.
Totipotent stem cells have the ability to differentiate into any kind of cell. They have the potential to create
a whole organism.
Pluripotent stem cells are very similar, however they can’t create the placenta or umbilical cord. Embryonic
stem cells (ESCs), cells in an embryo that is 3-5 days old (known as blastocyst at this stage), are all
pluripotent. Pluripotent cells cannot form an organism on their own.
Multipotent stem cells are limited to a family of related cells, ones for a specific tissue or organ. This also
relates to oligopotent stem cells, which is even more restricted to the types of cells it can transform into. Adult
stem cells, or stem cells that have the purpose of regenerating damaged tissue within an organism, are
multipotent
Dugesia Tigrina
Dugesia Tigrina is part of the dugesia genus of the planariidae family. Planariidae comes from the
order tricladida, which is part of the turbellarian flatworm class. It’s phylum
is platyhelminthes. Dugesia Tigrina is more commonly dubbed as “brown planaria”. The Latin roots of the term
“planaria” relate to the word planarius, or flat.
Dugesia Tigrina has many unique characteristics, apart from a complex stem cell system.
Brown planaria are scavengers, meaning they will typically eat dead organisms. They live in
freshwater environments, like ponds. Planaria is a bilaterally symmetrical organism, and are triploblastic,
meaning they have three germ layers (ectoderm, mesoderm, and endoderm.) Another main physical feature of
most planaria is that they are extremely sensitive to light due to eyespots that are capable of detecting light.
Scientists today are conducting behavioral experiments.
Stem Cell Structure and System in Dugesia Tigrina
This living structure has an astounding stem cell structure that is incredibly complex. Neoblast is a term
that describes the type of stem cell in the planaria, responsible for its speedy regeneration. When a flatworm
is amputated, the tissue cut off immediately regrows, due to these neoblasts, meaning that planaria
reproduce asexually. Neoblasts are pluripotent, meaning they have the ability to differentiate into any kind of
cell.
Correlation Between Oxidative Stress and Stem Cell Aging
Stem cell aging is the process where stem cells slowly start to degenerate, becoming weaker and losing
the ability to differentiate into different types of cells. Research has concluded that this process is due
to oxidative stress.
Oxidation is when a molecule loses an electron (molecule is called reductant), and transfers it to
another molecule (oxidant). These oxidants, or free radicals (oxygen split into individual atoms), are unstable due
to the odd number of electrons and become highly reactive. These free radicals then cause havoc to the cell
because they are stealing electrons, and create an imbalance between free radicals (associated with Reactive
Oxygen Species or ROS) and antioxidants. Antioxidants are substances that counter oxidation, creating a balance
that prevents oxidative stress, by giving up electrons to create an even number of electrons in the ROS
molecule. They neutralize free radicals. Studies have confirmed that oxidative stress is linked to primarily
neurological diseases, like Parkinson’s, Alzheimer’s, etc. It also has been linked to malignant (cancerous)
tumors.
The connection between antioxidants and stem cell growth is justified through the process of oxidation,
as negative effects, like stem cell aging, are caused by it. (I want to do more research with different
reaction mechanisms like HAT and SET, because they are extremely complex topics I have to do extensive
research on.) Antioxidants counter these effects, meaning that they also enhance stem cell differentiation
capability. Prominent antioxidants utilized in current trends include diferuloylmethane (curcumin), Coco
Nucifera (coconut oil), L-ascorbic acid (Vitamin C), alpha-tocopherol (Vitamin E), etc.
Examining the effect of antioxidants on dugesia tigrina will contribute to the development of
bioengineering since it is in regards to stem cell production, growth, and differentiation. It will also hold the key
to slowing down the aging process.
Research Goal
Observations, Results, and Conclusion
The planaria came in varying sizes, ranging from a few millimeters to 1-2 centimeters. According to the care-guide
provided with the organisms, the planaria cause the water to become cloudy after 1-2 days, however, after within the same
container, the water was still clear, and the planaria survived even after that time period mentioned in the care-guide.
Planarians have primarily contributed for regenerative medicine research, and behavioral
sciences research. Planarians can produce stem cells rapidly, and regrow amputated parts..
Antioxidants have a direct link to the growth of stem cells, as reactive oxidant species levels
can reduce stem cell proliferation. This oxidative stress has been linked to cardiovascular
disease (CVD), among other conditions. This experiment will test the effectiveness of
different antioxidants via the stem cell growth of dugesia tigrina.
Question: Which antioxidant is most effective at stimulating neoblast proliferation within
dugesia tigrina ?
Hypothesis: If the planaria are immersed in different antioxidant-water solutions, then the
planaria in the ascorbic acid solution will have the fastest growth rate, because of its
capability to lower LDL levels.
After amputation, I noticed that the piece cut off moved and squirmed for a few seconds. It was interesting to see how
its cells were still active even after amputation.
The planaria tested through each antioxidant and each trial had reacted in different ways. I noticed that the planaria
inhabiting the petri plates with the ascorbic acid had died only in the first 2-3 days, compared to the planaria inhabiting the
plates with the alpha-tocopherol and ergocalciferol.
I noticed minimal growth over the 2 week period, but there was error embedded in my procedure. The water evaporated in
most petri plates, due to that fact that I left it mostly uncovered, because of the fear that they would receive a lack of oxygen
if I fully enclosed the dish.
I had observed that mold had grown only in the petri dishes containing the ascorbic-acid solution, in all 3 trials. Perhaps this
was due to the fillers within the Vitamin C tablets, as the tablets weren't of pure vitamin-c.
Due to these unexpected complications, I wasn't able to collect accurate statistacal data, and the data was only able be
colllected from alpha-tocopherol and water. However, this doesn't defeat the scientific method, which is pure trial and
error. Hopefully, I'll be able to reconduct this experiment successfully by learning through these mistakes.
Variables and Methods
Variables:
Independent: Varying antioxidants (Vitamin C, D, and E)
Dependent: Growth rate of planaria over period of 2 weeks
Controls: Water without antioxidant concentration
Constants: Temperature, amount of water, amount of antioxidant, temperature, lighting, etc.
Materials:
•
Spring water (enough to fill 12 petri plates with 42.5 mL of water each)
•
Petri Plates (at least 12)
•
Pipettes
•
Gloves and necessary lab attire
•
Unit of brown planaria (supplied by Carolina Biological)
•
Microscope and slides
•
Plastic knives
•
Ruler with mm units
•
Vitamin C (21.25 mg/petri plate)
•
Vitamin D (21.25 mg/petri plate)
•
Vitamin E (21.25 mg/petri plate)
Bibliography
“Abstract Search.” Full Abstract,
abstracts.societyforscience.org/Home/FullAbstract?Category=Any Category&AllAbstracts=False&FairCountry=Any
Country&FairState=Any State&Keywords=stem cells&ProjectId=18166.
Cascalho, M, and J L Platt. “The Future of Organ Replacement: Needs, Potential Applications, and Obstacles to
Application.” Transplantation Proceedings, U.S. National Library of Medicine, Mar.
2006, www.ncbi.nlm.nih.gov/pmc/articles/PMC1475508/.
Deochand, Neil, et al. “Behavioral Research with Planaria.” Perspectives on Behavior Science, Springer International
Publishing, 9 Nov. 2018, www.ncbi.nlm.nih.gov/pmc/articles/PMC6701699/.
H;, Sisakhtnezhad S;Alimoradi E;Akrami. “External Factors Influencing Mesenchymal Stem Cell Fate in
Vitro.” European Journal of Cell Biology, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/27988106/.
“How Bone Marrow and Stem Cells Are Collected.” How Bone Marrow and Stem Cells Are Collected | BMT
Infonet, www.bmtinfonet.org/transplant-article/how-bone-marrow-and-stem-cells-are-collected.
Procedure:
Issigonis, Melanie. “Could We Use Planarians to Help Us Understand Human Regeneration?” Morgridge Institute for
Research, 7 Apr. 2021, morgridge.org/blue-sky/could-we-use-planarians-to-help-us-understand-humanregeneration/#:~:text=The stem cells that are,and tissues in the planarian.&text=Planarians have a
central nervous,regenerative capacity is very limited.
1. Prepare petri dishes and group each dish into categories for antioxidants (and control).
“Planaria (n.).” Etymology, www.etymonline.com/word/planaria#:~:text=Planaria (n.),Related: Planarian.
2. Reserve a tub of water for an environment for planaria to temporarily dwell in.
3. For each petri plate, make a solution of 42.5 mL of water to 21.25 mg of vitamin/antioxidant.
4. Transfer solutions to respective petri dishes.
5. Using pipette, transfer one organism per petri plate.
“Planaria: Psychology: George Fox University.” Psychology | George Fox
University, www.georgefox.edu/academics/undergrad/departments/psychology/ckoch/research/planimal.html#:~:text=
The eye spots are sensitive,taxis (change of direction).&text=Therefore, planarians can be conditioned with several
stimuli and responses.
“Regenerative Medicine.” Default, www.aabb.org/news-resources/resources/cellular-therapies/facts-about-cellulartherapies/regenerative-medicine.
6. Using a knife, amputate the flatworm by cutting it in the middle horizontally.
“Stem Cell Basics.” National Institutes of Health, U.S. Department of Health and Human
Services, stemcells.nih.gov/info/basics/stc-basics.
7. Record growth rate through observation and after 2 week period observe under microscope.
Measure through apparatus that has mm as units.
Yirka, Bob. “Researchers Discover Flatworms Retain Memories Even after Decapitation.” Phys.org, Phys.org, 12 July
2013, phys.org/news/2013-07-flat-worms-retain-memories-decapitation.html.
“Default - Stanford Children's Health.” Stanford Children's Health - Lucile Packard Children's Hospital
Stanford, www.stanfordchildrens.org/en/topic/default?id=what-are-stem-cells-160-38.
Additional photos
Antioxidant Models
https://www.worldofmolecules.com/
Ascorbic Acid
Alpha-Tocopherol
Ergocalciferol
ROS are oxygen containing molecules that have
an uneven amount of electrons, making them highly reactive, causing reactions known as oxidation.
These molecules (antioxidants) neutralize the effects of these free radicals by donating electrons.
Project Forms
Abstract