How Localization of Stem Cells Influences Rate of Regeneration in

Planaria (Flatworms)
Danielle Honan, Hannah Gorman, Lauren Bertelson, Kiera Jost

(planaria.jpg)

Background:

Planarians are flatworms, or simple creatures that

consist of three tissue layers: the ectoderm, mesoderm,
and endoderm, which are organized into organs and
organ systems. Common species of planaria are Dugesia
japonica (small brown), Schmidtea mediterranea (light
brown), Dugesia tigrinia (brown), Phagocata gracilis
(black), Dugesia dorotocephala (large brown), and
Procotyla fuviatilis (white). Most planarians live in
freshwater and can be found in ponds, streams, and under
rocks. They are (planar2.jpg) unique in that they possess a large number of adult stem cells,
which enables them to regenerate any part of their body, even the brain. Due to these
remarkable regenerative capabilities, planarians have become an exciting tool of study in stem
cell biology.
Link to short-documentary on planarian regeneration: https://www.youtube.com/watch?
v=roZeOBZAa2Q
With the development of new technologies, regenerative medicine has become a major
focus in scientific medical research. Many diseases, infections, and injuries could be treated
with the replacement of lost, damaged, or dysfunctional tissues. However, regeneration in
humans is limited, whereas many animals can regenerate body parts effectively. The planarian
is the champion of regeneration, and can regenerate any part of their body due to their
incredible stem cell system.
Various types of stem cells give rise to the specialized cells that carry out the bodys
specific functions, including muscle, skin, blood, and nerve cells. They can regenerate
themselves or produce specialized cells. These characteristics make them exciting in the field of
medicine because of the abundant possibilities to use them (F2.large.jpg) to create medical
treatments to replace lost or damaged cells.
Pluripotent stem cells are a type of stem cell
that have the ability to give rise to almost any
cell in the body. They have vast potential for
treatment of
disease as well
as generation of
cells/tissues for
transplants.
Pluripotent
neoblast cells
are found almost
everywhere in
the planarian
body (30% of all
cells in the adult
worm) and are responsible for the
incredible regeneration abilities of planarian. When a planaria loses a
body part, the neoblasts are reactivated and divide at the site of the
wound to form a blastema, which, through a complex process, allows
new complex structures to form. In humans, as well as many
mammals, pluripotency (ability of any one stem cell to produce
almost any cell type) only occurs in the early embryonic stage.
When planaria regenerate, the tail (regeneration in planaria(1).jpg)
regenerates a head and vice versa. However, if a planarian is cut in half only halfway through its
body, it can regenerate either two heads or two tails. There is no current understanding of how a
tail knows to produce a head or vice versa, though research is being done worldwide on
planarian. Research on planarian regeneration has (planarianstien.jpg) potential to help us
understand how stem cells work, though we are far from understanding how it works and how to
harness the potential.

In this lab, we are researching whether or not different cuts have any influence on the
time it takes for a planarian to regenerate. This will give us an idea about the distribution of stem
cells throughout the planarian body. The Sanchez Laboratory has published an interactive
diagram on stem cell lineage and markers in the planarian body. This data is promising in
understanding planarian regeneration as well as stem cells.
Link: http://planaria.stowers.org/stem_cell_lineage.php?category=1

Research Question: How does location of stem cells affect where planaria regenerate?
Where does it regenerate fastest? Does a cut site affect how long it takes to regenerate?
Are stem cells localized or spread throughout the body?

Hypothesis:
Stem cells that are spread out have to travel the same distance to begin growing
eyespots regardless of the cut. When planaria are cut into three sections, the different
sections will grow eyespots at the same time because the stem cells are equally
distributed throughout the planaria.

Procedure:
1) Label three petri dishes 1 through 3 and each fill half full with spring water. Set aside. (If
spring water is not readily available, fill a plastic container with a wide opening with tap water
and let sit overnight. This allows for the chlorine to evaporate which would otherwise kill the
worms.)

2) Transfer a planaria into each petri dish using a plastic transfer pipette.

3) In petri dish 1, cut the planarian as show in the corresponding chart(cut 1). Do the same for
the planaria in petri dish 2(cut 2), and 3(cut 3).

4) On the data form, record the time when finished cutting all of the planaria.

5) Make initial observations about the head fragments and tail fragment. Record them in the
areas indicated on the data form.

7) Every day, count the number of tail fragments in each dish that have regenerated
photoreceptors. Record the numbers with any other observations on the data form.

Variables:
Independent Different cuts (assigned and choice - we performed cut 3 and our
choice cut was to cut the tail in half vertically)
Dependent Growth (how fast it regenerates - measured by eye spots)
Controls specie of planaria (brown, Schmidtea mediterranea), temperature, light intensity

Data and Observations:

Day Time Cut # Number of fragments Observations

with photoreceptors

1 1:17 pm 3 1 of 2 Dark spots lining the cut site.

Light brown stuff coming out of
cut site. Both cut sites look the
same

1 1:17 pm choice 1 of 1 Black/dark brown on the edge of

the cut. Appears to be clear/light
brown substance coming out of
cut site.

2 1:32 pm 3 1 of 2 Cut tail looks slightly longer and is

a lighter gray. There is a dark spot
on the cut site.

2 1:32 pm choice 1 of 1 Tail chunk is curling into the other

part of the tail.

3 2:27 pm 3 1 or 2 Cut sites beginning to taper. Cut

off tail is beginning to move

3 2:27 pm choice 1 of 1 Edges of the cut are rounding off.

Tail still separate.

5 1:15 pm 3 1 of 2 The cut off tail is beginning to look

more tapered.both cut sites are
no longer blunt.

5 1:15 pm choice 1 of 1 The cut site looks like it is healing,

but the tail chunk is still separate
from the tail.

8 12:45 pm 3 1 of 2 The original worm looks fully

healed. On the tail it looks like 2
photoreceptors are beginning to
form.

8 12:25 pm choice 1 of 1 The tail chink is still separate,

moving, and curled towards the
body
9 1:05 pm 3 2 of 2 The cut tail now has 2
photoreceptors and cut looks
healed.

9 1:05 pm choice 1 of 1 The chunk of tail is moving, and

shaped like a hook.

Pictures:

Choice Cut Day 1:

Cut 3 Body Day 1:
Cut 3 Tail Day 1:
Choice Cut Day 2:

Cut 3 Body Day 2: Cut 3 Tail Day 2:

Choice Cut Last Day: Cut 3 Tail Last Day:

Data Analysis:

T-Test: the difference between the averages of data

pairs (Ex. between Cut 2 and cut 3). This results in a P-value
(P-value between Cut 2 and 3 = .7701) To have a 95%
confidence, meaning the p-value proves correlation between
the data points with 95% certainty, the p-value must be less
than or equal to .05. All of our data is significantly larger than .
05 meaning there is only weak evidence prove correlation. Our data is lower than the
maximum, 4.3, any data point should be with 3 study groups and 95% confidence. This
means the experiment wasnt severely messed up. Once the null hypothesis is weakly
proven to be false, the next thing to be determined is whether the data proves the stem
cells are localized or not. If the stem cells are localized, there would be variation between
the number of days it took for the planeria to grow eyes. The cut closest to the localized
stem cells would grow eyespots significantly earlier than the others. The other possibility
is that the stem cells are spread out. This would be indicated by eyespots on the planaria
fragments appearing at roughly the same time. If the stem cells are spread out, they will all
reach the cut close to the same time, and eyespots will form at close to the same time. The
data indicates that the stem cells are spread out. The average for cut one was 8.75 days,
the average for cut two was 8.4 days, and the average for cut three was 8.5. These
averages are all within less than a day of each other, meaning the stem cells in planaria are
spread through the body.

Conclusion Links:
Danielle:
https://docs.google.com/a/bvsd.org/document/d/1o1ovgLiuNxvTp1j1VeAMCJGNxZ2t6Ls-
v64h2yuFnaw/edit?usp=sharing
Lauren:
https://docs.google.com/a/bvsd.org/document/d/1xknuRhdrkmgAxyTTpJwtydHGxHkd8BdBuY4z
qFBrgN4/edit?usp=sharing

Hannah:
https://docs.google.com/a/bvsd.org/document/d/13TLlUyUpqRzEvd1WIKfrEOAr9UILjqg36yAEQ
4GNr68/edit?usp=sharing

Kiera:
https://docs.google.com/document/d/1c4sNqlT9lwh5t19pmW_R0Rj8VQcz0QbCBP11d1cywRw/
edit

Contributions:
Danielle: Background information & pictures in background, Research Question, Variables,
Works Cited
Lauren: Procedure, Pictures of the planaria, Data and Observations
Hannah: Analysis, Hypothesis, graphs/tables, p values, t-test
Kiera: Procedure, Hypothesis, graphs/pictures, Observations

Works Cited

Alvarado, Alejandro Snchez, Sofia Robb, and Eric Ross. "Stem Cell Lineage." The

Snchez Laboratory - Regeneration Research. Stowers Institute for Medical Research,

n.d. Web. 08 Feb. 2017. <http://planaria.stowers.org/stem_cell_lineage.php?category=2>.

Bartscherer, Kerstin. "Flatworms, the Masters of Regeneration but Nothing Can

Happen without Stem Cells." Cell Biology, Developmental Biology. Max - Planck -

Gesellschaft, 3 June 2014. Web. 08 Feb. 2017. <https://www.mpg.de/8244494/flatworms-

regeneration>.

Gentile, Luca, Francesc Cebri, and Kerstin Bartscherer. "The Planarian Flatworm: An in

Vivo Model for Stem Cell Biology and Nervous System Regeneration | Disease Models

& Mechanisms." Disease Models & Mechanisms. The Company of Biologists, 19 Dec.

2011. Web. 08 Feb. 2017. <http://dmm.biologists.org/content/4/1/12>.

Rink, Jochen C. Stem Cell Systems and Regeneration in Planaria. Development Genes

and Evolution 223.1-2 (2013): 6784. PMC. Web. 8 Feb. 2017.

<https://www.ncbi.nlm.nih.gov/pubmed/23138344>

Unknown. "Introduction to Planaria." Introduction to Planaria (n.d.): n. pag. MIT

Department of Biology. MIT. Web. 8 Feb. 2017.

<https://biology.mit.edu/sites/default/files/Introduction%20to%20Planaria(1).pdf>.

Unknown. "Planaria: A Window on Regeneration." Exploratorium. N/a, n.d. Web. 8 Feb.

2017.

<http://www.exploratorium.edu/files/imaging_station/research/planaria/story_planaria.pd

f+>.