3D BIOPRINTING & BIOFABRICATION
Laura Maria Gonzalez
May 11, 2021
Human induced pluripotent stem cells (hiPSCs) mimic human embryonic stem cells in their ability to differentiate into almost any cell type, but are
generated from somatic cells. This week we tested various drugs on George Church's own hiPSC line that was differentiated into cardiac organoids
named Engineered Heart Tissues (EHTs). The organoids are 3D tissue-like structures comprised of multiple cell types which originate from stem
cells. They are able to undergo self-organization and can mimic the architecture and functionality of in vivo organs which means they could be
used to assess effects of drugs or viruses like SARS-CoV2 in vivo settings.
Hypothesis of expected effects of the following drugs on the heart:
For the study I chose theobromine. It is notably found in cocoa and is thought to have "pleasurable" effects at low levels while causing anxiety at
high levels. Studies show that increasing levels of Theobromine dose-dependently increased heart rate. I was not able to find studies that looked
into specific concentrations of theobromine, but found sources noting that it can have effects similar to about 1/10th of caffeine. Studies typically
used amounts from 250mg-1000mg pills given daily to participants. Consulting with the TA I landed on an initial concentration of 100uM. It seems
like we will be shifting the experiment to test various dilutions so I am interested to see if the effects are dose-dependent and how theobromine
compares to caffeine.
Caffeine - Increase heart contraction rate
Oxytocin - Slows the heart rate and decreases cardiac contractions, dose dependent
Atosiban - Used as a labour repressant to halt premature labor. No effect on heart contraction rate vs. a control is expected
Isoprenaline - Increase contractility
Theobromine - Related to caffeine, found in cocoa at high levels, dose dependently increase contraction rate
Adrenaline - Released during stressful conditions. Increases heart contraction rate
Norephinephrine - Released during stressful conditions. Increases heart contraction rate
Pre-Lab Questions:
From an ethical and moral standpoint, list pros and cons of working with HiPSCs as opposed to embryonic stem cells?
Embryonic stem cells are cells from which other cells with specialized functions can be generated. They can divide to form either more stem cells or
specialized cells. This property makes them desirable because they can be used to regenerate and repair diseased or damaged tissues or test
drugs for safety and effectiveness. While offering desirable properties, the use of embryonic stem cells is controversial because they come from
human embryos. Although adult human stem cells can be used, they are not as versatile or durable.
HiPSCs offer a promising alternative since they can mimic human embryonic stem cells by differentiating into almost any cell type, but are
generated by somatic cells (fibroblast skin cells) through reprogramming. This has the potential to remove the need to use embryonic stem cells and
also offers the advantage of being sourced from individual patients for personalized medicine and study of drug safety and effectiveness. The
downside to HiPSCs is reports of variability such as reduced or variable yields of neural and cardiovascular cells.
If we are to use organoid models as surrogates for in vivo tissues, what type of functional attributes would be critical to add/engineer into such
organoid systems?
Some critical attributes to add/engineer into such organoid systems might be co-cultures. For example if you are to generate gut organoids it may
be unproductive to study without their respective microbiome. Also finding ways to test and study impacts of immune cell populations within
organoids.
Can you think of any limitations in current organoid and/or organ on a chip systems in recapitulating human biology?
I oftentimes wonder about the interconnectedness of the body and our environment. How many toxins does our body encounter and interact with?
What is the impact of diet or symbiotic bacteria on our living systems. Managing complexity with organoids and chip systems seems to be a
limitation when trying to address these factors.
If you were to theorize an experiment about improving current organoid models, which aspects of the microenvironment would you consider adding
beyond chemical?
Mechanical aspects such as vibration? Our bodies move through space and time therefore not only are organs moving rhythmically, but also
through our external actions - activities, sports. I think it would be interesting to develop an experiment that include external vibrations or
movements on organoids such as the heart or gut.
In the tissue engineering paradigm, what properties are important to provide from a scaffold and bioreactor?
From the scaffold it is important to provide porosity to allow for vascularization therefore the composition, architecture, and mechanical properties
should be preserved. Properties that are key to provide from a bioreactor are environmental control and exchange of nutrients, oxygen, and
metabolites.
PART A: HIPSC
EHT DRUG TESTING
THEOBROMINE
Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs), figure provided in class
Mycelium Running, Paul Stamets
Peat Seed Starter Trays, Amazon
Top View
Sloped Top and Bottom Faces
Placed in the Ground
Side View
Seedling Mycorrhizal Network, Root Rescue
One of my favorite take away from Paul Stamets "Mycelium Running" is the idea of a bioengineering science dedicated to programming myco-
neurological networks that monitor environmental health and threats. Stamets describes how the networks sense factors from footsteps to falling
tree branches and could relay information about the movements of all organisms through the landscape.
Suzanne Simard also emphasizes the impact of mycorrhizal networks on old growth forests and it's impact on nutrient flow where mother trees pass
carbon, nutrients, and water through the roots to adjacent seedlings.
Attempting to think about what the mycelium would want and with these thoughts in mind I thought about a design that could extend the
mycorrhizal networks. To do so I thought about designing seedling pots / germination containers. These pots are typically made of peat and used to
prevent transplant shock. I thought it would be interesting to design these out of mycelium that could then be introduced into the soil to foster
seedling root networks. Design factors to explore may include the effect of mycelium geometry on the growth and development of roots.
PART B: MYCELIUM
Narsinh, Kazim H., Jordan Plews, and Joseph C. Wu. "Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical
twins?." Molecular Therapy 19.4 (2011): 635-638.
Kim, Jihoon, Bon-Kyoung Koo, and Juergen A. Knoblich. "Human organoids: model systems for human biology and medicine." Nature Reviews
Molecular Cell Biology 21.10 (2020): 571-584.
Bhumiratana, Sarindr, et al. "Tissue-engineered autologous grafts for facial bone reconstruction." Science translational medicine 8.343 (2016):
343ra83-343ra83.
Baggott, Matthew J., et al. "Psychopharmacology of theobromine in healthy volunteers." Psychopharmacology 228.1 (2013): 109-118.
REFERENCES
No Addition of Theobromine
10uM of Theobromine