1. The document discusses advances in stem cell and tissue engineering research that enable the generation of complex biological entities, including synthetic human embryo-like entities (SHELEs). 2. These new methods raise ethical concerns similar to those regarding human embryo research. Current guidelines, including the 14-day rule, do not provide clear guidance on research involving SHELEs. 3. The authors propose that ethical boundaries for this type of research should be based on the direct features that indicate moral status, such as the biological substrates for pain perception, rather than pre-emptive stopping points like the 14-day rule that can be bypassed through technological progress.

1. Stem cells, engineered tissues, and synthetic embryo-like entities:
State of the science and ethical implications
George Church & John Aach
Ethics of Early Embryo Research & the Future of the 14-Day Rule
Harvard
November 7, 2016
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2. Take-homes
1. New methods in hPSC* and tissue engineering are rapidly
enabling generation of complex biological entities including
Synthetic Human Embryo-Like Entities (SHELEs)
2. SHELEs are promising tools for research into human development
but raise ethical concerns like those raised by embryo research
3. These challenges relate to the 14-day rule but cannot be met
simply by adjusting the 14 day rule
4. Therefore, ethical boundaries for research need to be based as
directly as possible on features that signify moral status vs.
embryonic features that usually precede their appearance
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* hPSC = human Pluripotent Stem Cell = hESC or hiPSC
hESC = human Embryonic Stem Cell
hiPSC = human induced Pluripotent Stem Cell

3. Acknowledgements
Scientific
• Church lab (present): John Aach, Alex Ng, Eswar Iyer,
also Evan Appleton, Alex Chavez, Jeremy Huang, Parastoo Khoshaklagh, Wren Saylor,
Seth Shipman
• Church lab (former): Volker Busskamp, Susan Byrne, Nathan Lewis, Prashant
Mali, Luhan Yang
• Jennifer Lewis & lab: Mark Scott
• Ron Weiss & lab: Patrick Guye
Ethics
• Paper in revision with John Aach, Jeantine Lunshof, Eswar Iyer
– Acknowledging: George Q. Daley, M. William Lensch, Robert D. Truog, Dan I.
Wikler, I. Glenn Cohen, J. Hyuk Lee, Catherine Spina, James J. Collins, and Henry T.
Greely
Funding
• NIH grants RM1HG008525 and P50HG005550
• People Programme (Marie Curie Actions) of the EU’s 7th Framework 665
Programme (FP7/2007-2013), REA grant agreement n°298698
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4. Key directions in human embryo and pluripotent stem cell research
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hPSC
Direct differentiation
to diverse cell types
DAPI
DAPI
DAPI
neuronmuscleendothelial
Develop organoids
from embryoid bodies
cerebralgastric
many
others…
• https://embryology.med.unsw.edu.au/em
bryology/images/9/98/Human_embryo_da
y_5.jpg
• Gist Croft, Cecilia Pelligrini, Ali H.
Brivanlou, Rockefeller University
Human embryos
5d blastocyst
12d embryo
Engineered hPSC-based
tissues & organoids
cardiomyocytes
microcephaly
Alex Ng et.al. (unpublished); Lancaster et.al. (2013) Nature 501:373,; McKracken et.al. (2014)
Nature 516:400; Wang et.al. (2014) Nat Medicine 20:616, Mark Scott et.al. (unpublished)

5. Ways to make hPSC generate specific cell types or phenotypes
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Add factors to media
hPSC
• Special factors needed
for almost all conversions
• Factors also needed to
maintain pluripotency
Transcription
Factor
hPSC
Express transcription factors
• Exogenous TFs deliverable via
lentiviruses &c, often many at
once
• Endogenous copies of TFs can
also be induced (e.g., via dCas9)
• TFs can be inducibly expressed
either way
hPSC

6. Conversion of hiPSC to neurons: Church lab experience
We have worked with both expression of exogenous TFs and Cas9 as ways of
inducing conversion of hiPSC to neurons. TFs have proved more efficient.
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Expression of
exogenous TFs
doxycyline
inducible
NGN1 & NGN2
hPSC
dCas9 induction
endogenous TFs NGN2
hPSC
doxycyline
inducible
DAPI
-III Tubulin
METHOD CONVERSION YIELD
90%
7%
Busskamp (2014) Mol Syst Biol 10:760; Chavez (2015) Nat Methods 12:326

7. Currently using high throughput method for generating cell types
using complete set of human TFs
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• Human TFome: First complete collection of human TF constructs
– Assembled from external collections* + gene synthesis
• Method: Infect hiPSC with lentivirus TFome library, induce, and screen for loss of
pluripotency; then identify derived cell type
• We have identified ~100 TFs that lead to loss of pluripotency and are now classifying
the cell types generated by them.
* Human ORFeome (Vidal lab) + Taipale lab + LaBaer lab

8. Rapid generation of neurons, myocytes, and endothelium using TFs
identified by TFome screen
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4 day TF induction

9. Ways of engineering tissues and organoids
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Culturing regimes
(including 3D culture)
Microfabrication
3D printing
Cerebral
organoid
Cardiochip
Renal
tubule
Lancaster (2013) Nature 501:373; McCain
(2014) Biomaterials 35:5462; Homan
(2016) Sci Rep 6:34845

10. Combining hPSC genetic engineering with organoid engineering to
generate vascularized organoids
• Lack of vasculature in organoids limits their growth, development, &
complexity
• We are collaborating with Jennifer Lewis on generating vascularizing cerebral
organoids
• Goal: Combine Lewis lab methods for engineering tissues with perfusable
vasculature with our methods of generating cell types from engineered hPSC
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necrotic
core
Lancaster (2013)
Nature 501:373

11. Example of Lewis lab engineered vascularized tissue construct
Channels of cell-laden or ‘fugitive’ gels
are printed and covered with gel matrix.
Fugitive gels then evacuated & lined with
endothelial cells to form vasculature.
Image of 3D printing of the channels.
Vascularized channels can be hooked up
to external perfusion pumps.
Finished construct. Each of the 3 cell
lines laid down fluoresces with different
color.
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Kolesky (2104) Adv Mater 26:3124

12. We are getting close to generating cerebrovascular organoids
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Organoid exhibiting layers of neural types
and endothelial cells
Lumen formation in hiPSC-derived
endothelium
Images courtesy of Mark Scott

13. Next steps
• Encourage cerebrovascular organoid to sprout vessels into gel medium and
connect to pin-casted vasculature and external perfusion apparatus.
• Augment the cerebrovascular organoid with additional cell types (e.g., glia,
pericytes, …)
• Refine hiPSC differentiation and protocols to better match brain-specific
subtypes such as Brain Microvascular Endothelial Cells and model BBB
13Images courtesy of Mark Scott

14. Ethical implications
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15. Why the rapid advances in Stem cell/Organoid engineering will
raise new ethical challenges
1. Vascularization and tissue engineering will have wide application.
– Vascularized Embryoid Bodies = obvious next step; will lead to more complex
Synthetic Human Embryo-like Entities (SHELEs)
2. We are also becoming adept at generating artificial tissues comprising
multiple germ layer lineages and that can continue to develop.
– SHELEs don’t have to be anything like intact human embryos
3. These SHELEs will be useful for research but may excite moral concerns
similar to those raised by embryos.
4. Current embryo and stem cell ethical rules for research give little guidance
on SHELEs and are not structured in a way that can readily extend to them
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16. The Warmflash experiments give an early taste of what is to come
• Microfabricated hPSC colonies induced to form an apparent Primitive Streak
• Recognized as not in violation of 14 day rule and as useful research tool, but
calls attention to ambiguities/inconsistencies in related ethical rules & laws*
• ISSCR’s 2016 guidelines include recommendations for review of experiments
on organized embryo-like cellular structures that might manifest human
organismal potential; prohibition if they develop a Primitive Streak†
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* Denker (2014) Cells Tissues Organs 199:221; Pera et.al. (2015) Nat Methods 12:917; Hyun et.al. (2016) Nature 533:169
† ISSCR (2016) Guidelines for Stem Cell Science and Clinical Translation, sections 2.1.3.2, 2.1.3.3, Glossary entry Embryo-like structures
Warmflash (2014) Nat Methods 11:847

17. Why adjusting the 14 day rule will not solve the fundamental problem!
• The 14 day rule is supposed to stop the experiment before it gets to a morally
concerning state
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Images: http://i.stack.imgur.com/lkX4a.png , Larsen’s Human Embryology Fig. 4-7
• hPSC/tissue engineering
could allow us to bypass
the PS and directly create a
neurulating SHELE
• In like manner, any pre-emptive stopping point could potentially be
bypassed using advanced hPSC/tissue engineering
Neurulation: beginning of
brain formation allows
possibility of pain
PS

18. Basing research limits on features of direct moral concern
vs. pre-emptive stopping points
If stopping points like the PS are ineffective because they can be bypassed, how
can workable ethical boundaries for SHELE research be formulated?
Our proposal
1. Avoid basing boundaries on canonical features / stages of embryos, and
base them on the biology that directly underlies features of moral concern.
– Example: If possibility of experiencing pain a moral concern, define stopping point
at appearance of biological substrates for pain perception
2. Comprehensively consider and evaluate all features of moral concern, not
just the first canonically occurring one.
– Biological substrates of pain could be ~20 wks*, but other features may be morally
concerning too (acquisition of heartbeat, human form,…)†. All must be considered.
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We don’t underestimate the difficulty of this proposal, but see no other choice if
potentially huge benefits of using hPSC/organoid engineering to understand
human development are to be explored and realized.
* Gestational age. Refs: Lee (2005) JAMA 294:297, Lowery (2007) Semin Perinatol 31:275
† NIH (1994) Report of the Human Embryo Research Panel, I, chap. 3 pp. 37-8, 46-7

19. Why current embryo/stem cell research guidelines are
unprepared for SHELEs
• Where guidelines concerned with development, have taken canonical
pathway from pluri/toti-potent cell embryo fetus as conceptual model
– Recall guidelines’ history: IVF/ART (1970s) hESC /cloning (2000s) …
• Most rules concerned with development limit use of methods that could
activate full developmental program or allow it to proceed
• New hPSC / tissue engineering methods are revealing unexpected plasticity
of development and achieving non-canonical results
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recognize
Result: Current guidelines …
don’t recognize

20. Research into human development involving embryos and SHELEs:
Contrasting concepts of ethical guidelines
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PS
Current guidelines
• Development a single highway
taking hPSC embryo fetus
• Restrictions are STOP signs
restricting passage on the highway
Our proposal
• Development a main road, but off-
road travel possible with
hPSC/tissue engineering
• Restrictions are perimeter fences
restricting entry to areas of moral
concern

21. Efforts for devising ethical guidance for SHELE research, and for re-
evaluating of the 14 day rule, should aim for consistency
• Both must revisit question of what features of developing embryo signify
emergence of an entity we are morally obliged to spare from experimentation
– If PS and day 14 deemed not of this sort, what features are? These would need to
be considered in SHELEs as well as embryos
– But this would only be starting point for devising rules for SHELEs …
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• Ideally we should aim for integrated guidelines for embryos, hPSC, and SHELEs

22. Our recommendation
• A wide-ranging effort should be organized to explore whether and how far
embryo/stem cell research guidelines can be revised to
– base them more directly on morally concerning features
– take SHELEs and hPSC/tissue engineering methods into account.
• This effort should be
– international
– jointly involve scientists, bioethicists, policy makers, and other concerned parties
– be coordinated with other efforts looking to re-evaluate the 14 day rule.
• Ultimately we would like to see this effort result in a commission that will
revise and approve ethics guidelines that cover research involving stem cells,
embryos, and SHELEs.
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23. Thank you
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24. Extra slides
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25. Nociception and Pain development
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Lee (2005) JAMA 294:297

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