What are Embryonic and Adult stem cells?
Stem Cell and Organ Regeneration
Alice Basin
Daiji Kano
Experimental Cell and Developmental Biology 03-345
Section B
04/13/2010
Embryonic Stem (ES) Cells
Undifferentiated cells derived
from the inner cell mass (ICM) of
the blastocyst stage embryos.
Are capable of indefinite self
renewal in culture: (applies to
Mouse ES cells) supplied with LIF
(Leukemia Inhibitory Factor)
belongs to IL-6 cytokine family. It
binds to its gp130 receptor and
leads to activation of differentiating
and anti-differentiating signals at
the same time.
Maintain normal karyotpe,
pluripotent and have the ability to
differentiate into multiple cell types
of the three germ layers.
Google Images: ://theblackcordelias.files.wordpress.com/2009/03/stem-cells.jpg
Embryonic Stem Cells; human vs.
mouse
•
LIF can replace the requirement for
feeder cells and serum entirely for
mouse ES cells (mESCs)but not for
human ES cells (hESCs). hESCs have
the ability to form trophoblast cells in
response to bone morphogenetic
proteins, mESCs do not.
•
hESCs and mESCs differ in the
expression of several cell surface
antigens.
•
Have the ability to treat degenerative
diseases and major traumatic injuries,
which may result in a significant
improvement in the quality and length
of life for affected patients.
•
Can be used as a model for early
human embryonic development
•
http://www.sciencemag.org/cgi/content/full/282/5391/1145/F4
Teratomas formed by the human ES cell lines in SCIDbeige mice. Human ES cells after 4 to 5 months of culture
Figure on the right shows that when from about 50% confluent six-well plates were injected into
ES cells were injected into immuno
the rear leg muscles of 4-week-old male SCID-beige mice.
compromised mice, teratomas of
Seven to eight weeks after injection, the resulting
muliple cell types formed.
teratomas were examined histologically
Adult Stem Cells
•
Tissue specific, only able to give rise to progeny cells
corresponding to their tissue of origin.
•
In contrast to ES cells, immature versions of the cells normally
found in the tissue from which they were removed and so are
usually only able to differentiate into mature cells of the same or
similar type.
•
Similar to ES cells, adult stem cells also have the ability to divide
or self-renew indefinitely, and generate all the cell types of the
organ from which they originate.
•
Unlike ES cells, the use of adult stem cells in research and
therapy is considered to be less of an ethical concern as they are
derived from adult tissue samples rather than destroyed human
embryos.
Stem Cells and Organ
Regeneration
Stem cells and organ regeneration
•
•
Liver is the only organ in body
capable of regeneration
Organ transplantation:
– Need for a donor: availability is
decreasing
– Demands that specific conditions
are met such as blood type
– Complicated process (surgically and
emotionally)
• Major surgeries often come with
great risks
• Are people comfortable with being
n% pork?
•
•
What else can we do?
Stem cell mediated organ
regeneration
http://www.ptei.org/assets/METHOD_ARM.jpg
Stem cells and organ regeneration
• How do stem cells contribute to organ regeneration?
– Adult cardiac stem cells are multipotent and support
myocardial regeneration
– Mesenchymal stem cells (MSC) differentiate into bone,
cartilage, and fat cells
– Hematopoietic stem cells (HSC) contribute to the
regeneration of renal tubules after renal ischemiareperfusion injuries
Stem cells and organ regeneration
•
Can take the form of:
– Scaffold transplantation: Stem cells are
grown on a 3D scaffold. The cells start
secreting growth factors and form a living
tissue, which is then transplanted in the
patient. The scaffold biodegrades as the
stem cells differentiate to repair the organ.
2D->3D = better mimics actual organ and
therefore more efficient
– Hematopoetic Stem Cell (HSC)
Mobilization: mobilization of the
multipotent stem cell that give rise to all
the blood cell types to the site of action,
followed by differentiation at the target
location
– HSC injection: similar to HSC mobilization
– MSC (Mesenchymal Stem Cell): often
used in adjunction with stem cell
transplantation. Can differentiate into
osteoblasts, (bone cells), chondrocytes
(cartilage cells) and adipocytes (fat cells)
http://www.biomaterials.org/week/bio25_clip_image003.jpg
http://www.sigmaaldrich.com/etc/medialib/life-science/stem-cellbiology/mesenchymal-stem-cell.Par.0001.Image.457.gif
Stem cells and organ regeneration
• Advantages over the conventional organ transplantation:
– Availability and feasibility
– Autografting and isografting
• Still a controversial topic
– long-term or permanent presence of foreign cells in the recipient, i.e.,
cells that cannot be retrieved
• tumor formation
• fibrosis
Final Project
Our project
• Project Goal: to examine the effect of caffeine
treatment on myoblast differentiation.
• Hypothesis: If caffeine is added to myoblasts
and myotube stages of C2C12 cells, then this
will cause an increase in intracellular calcium
from intracellular stores which will accelerate
myogenesis.
Our project
• Details of what we are going to test:
1. We are going to test whether or not caffeine causes the increase
of intracellular calcium and if that effects cell differentiation.
2. We are going to have cells in NES, containing EGTA (calcium
chelator) in order to confirm whether or not the presence of [Ca2+
] ex is necessary for myogenesis.
3. [Ca2+]in will be monitored by Fura-2 tagging of [Ca2+]in.
– Fura-2 is a calcium ion chelator that fluoresces in the UV range
• Used as an indicator of [Ca2+] fluctuation
Protocol for treatment of cells
We will be treating 7 dishes of cells in total:
• Day 0 (Monday)
– Cells to be proliferated only (no differentiation)
1. Control plate with no treatment
2. Plate with 20mM Caffeine treatment
– Cells to be differentiated on Day 2 (Thursday)
3. Control plate with no treatment
4. Plate with 2mM Caffeine treatment
5. Plate with 20mM Caffeine treatment
• Day 2 (Thursday)
– Cells whose [Ca2+]in will be observed by Fura-2
treatment
6. Control plate with no treatment
7. Plate with 20mM Caffeine treatment
– Might be a different concentration depending on observations from Lab
Day 2
Current Research related to Final Project:
“Caffeine and Nicotine decrease acetylcholine receptor clustering
C2C12 myotube culture”
• Experimental Goals: assess the effects of short-term and long-term
exposure to caffeine and nicotine on the frequency of AChR
clustering on myotubes and possibly on myogenesis.
• Hypothesis: Physiologically significant concentrations of caffeine
would decrease AChR clustering, and that combinations of caffeine
and nicotine would decrease AChR clustering beyond the effect of
either treatment alone.
Methods:
•
Short term treatment: C2C12 cell cultures were maintained as untreated controls or
exposed to caffeine, nicotine, or caffeine and nicotine during the last 48 h of 72 h in
Differentiation Media (DM) . Caffeine concentrations from1mM-1 nM were tested for
the ability to decrease agrin-induced and spontaneous AChR clustering, with 10 μM
caffeine being demonstrated as sufficient to do so.
•
Long term treatment :C2C12 cell cultures were maintained initially in10-cm plates
as untreated controls or exposed to 10 μM caffeine, 1 μM nicotine, or 10 μM caffeine
and 1 μM nicotine in Growth media (GM) for several generations over 2 weeks.
Treatments continued when cultures were transferred to cover-slips and then for 72 h
in DM.
•
AChR clustering assay: C2C12 cell cultures were maintained in six-well plates with
experimental manipulations as described above. AChRs were labeled by the binding
of α-bungarotoxin conjugated to tetramethyl rhodamine. Cultures were incubated in
the toxin-containing medium for 30 min at 37°C to label AChRs.
Data/Results
• “When exposed to
physiologically significant amount
of caffeine, C2C12 myotubes
cluster AChRs in response to agrin
at a decreased frequency when
compared with untreated culture”
Fig. 3 C2C12 myotubes were viewed and photographed by
fluorescence microscopy after 72 h in DM, with exposure to caffeine
and/or nicotine for the last 48 h in DM and to agrin for the last 16 h
in DM. a With 10 ng/ml agrin. b With 10 μM caffeine and 10 ng/ml
agrin.
Image source: KordorskyHerrrera et al
Fig. 1 Caffeine decreases the frequency of agrin-induced AChR
clustering with a dose response. AChR clusters were determined for
C2C12 myotube cultures treated for 48 h in DM with various levels of
caffeine and for the final 16 h with 10 ng/ml agrin. Each column
represents the average for 25 fields of view (error bars SEM).
*Statistically decreased in comparison with control, at P<0.01 with
Student’s t-test
Discussion/Conclusion
• Caffeine (as well as nicotine) decreases AchR clustering
• Intracellular calcium is known to be involved in agrindependent Ach aggregation
• Caffeine may impact calcium in a similar way calcium
chelators like BAPTA does—by depleting intracellular
calcium stores—and therefore interfere with calciumdependent agrin-induced synapse formation
References
1. Wobus, Anna M.: Google Books: Stem Cells: Handbook of experimental pharmoclolgy, Springer
2. Thompson, James A. et al. “Embryonic Stem Cells derived from human
blastocysts:http://www.sciencemag.org/cgi/content/full/282/5391/1145 Science AAAS
3. Stemagen: Stem Cell Research and development:Stem Cell Overview:
http://www.stemagen.com/overview.htm
4. Van Hoof et al. Molecular and Cellular Proteomics: “A quest fo human and mouse embryonic stem
cell-specific proteins
5. Kordorsky-Herrrera et al. Cell Tissue Res. “Caffeine and Nicotene decrease acetylcholine receptor
clustering in C2C12 mouse myotube culture” (2009) Springer-Verlag
6. Levenberg, S. et al. Differentiation of human embryonic stem cells on three-dimensional polymer
scaffolds. Proc. Natl. Acad. Sci. USA 100, 12741–12746 (2003).