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:
DataAnalysis:

T-Test:thedifferencebetweentheaveragesofdata
pairs(Ex.betweenCut2andcut3).ThisresultsinaP-value
(P-valuebetweenCut2and3=.7701)Tohavea95%
confidence,meaningthep-valueprovescorrelationbetween
thedatapointswith95%certainty,thep-valuemustbeless
thanorequalto.05.Allofourdataissignificantlylargerthan.05meaningthereisonly
weakevidenceprovecorrelation.Ourdataislowerthanthemaximum,4.3,anydatapoint
shouldbewith3studygroupsand95%confidence.Thismeanstheexperimentwasnt
severelymessedup.Oncethenullhypothesisisweaklyproventobefalse,thenextthing
tobedeterminediswhetherthedataprovesthestemcellsarelocalizedornot.Ifthestem
cellsarelocalized,therewouldbevariationbetweenthenumberofdaysittookforthe
planeriatogroweyes.Thecutclosesttothelocalizedstemcellswouldgroweyespots
significantlyearlierthantheothers.Theotherpossibilityisthatthestemcellsarespread
out.Thiswouldbeindicatedbyeyespotsontheplanariafragmentsappearingatroughly
thesametime.Ifthestemcellsarespreadout,theywillallreachthecutclosetothesame
time,andeyespotswillformatclosetothesametime.Thedataindicatesthatthestem
cellsarespreadout.Theaverageforcutonewas8.75days,theaverageforcuttwowas
8.4days,andtheaverageforcutthreewas8.5.Theseaveragesareallwithinlessthana
dayofeachother,meaningthestemcellsinplanariaarespreadthroughthebody.

Conclusion Links:
Danielle:
https://docs.google.com/a/bvsd.org/document/d/1o1ovgLiuNxvTp1j1VeAMCJGNxZ2t6Ls-v64h2
yuFnaw/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+>.