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George Church and John Aach, "Stem Cells, Engineered Tissues, and Synthetic Embryo-Like Entities"


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November 7, 2016

For over 35 years, the “14-Day Rule,” prohibiting in vitro experimentation on embryos beyond 14 days, stood as an ethical line in the sand for embryo research around the world. Throughout the arc of the rule’s existence it had not been questioned, as scientists had been unable to grow embryos in vitro either up to, or beyond, 14 days; a practical limitation that served as a backstop to the ethical rule. However, in May 2016, labs in the U.S. and the U.K. were the first to report being able to sustain human embryos in vitro for up to 13 days. This development and other advances in in vitro research involving organized, embryo-like cellular structures have raised a number of questions about the rule, its genesis, application, and future scope. This conference convened experts in bioethics, stem cell research, embryology, and law to discuss the ethical underpinnings and future scope of the rule. Questions discussed included:

- What are the historical, ethical and scientific rationales for establishing the 14-Day Rule?

- Should the 14-Day Rule be revisited in light of recent advances?

- Should the 14-Day Rule even apply to research involving the in vitro culture of embryo-like cellular structures?

This event was free and open to the public.

This event was sponsored by the Harvard University Office of the Vice Provost for Research, the Edmond J. Safra Center for Ethics at Harvard University, and the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School, and the Harvard Stem Cell Institute, with support from the International Society for Stem Cell Research and the Center for Bioethics at Harvard Medical School.

View the full agenda and learn more on the website:

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George Church and John Aach, "Stem Cells, Engineered Tissues, and Synthetic Embryo-Like Entities"

  1. 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 1
  2. 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 2 * hPSC = human Pluripotent Stem Cell = hESC or hiPSC hESC = human Embryonic Stem Cell hiPSC = human induced Pluripotent Stem Cell
  3. 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 3
  4. 4. Key directions in human embryo and pluripotent stem cell research 4 hPSC Direct differentiation to diverse cell types DAPI DAPI DAPI neuronmuscleendothelial Develop organoids from embryoid bodies cerebralgastric many others… • 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 (unpublished); Lancaster (2013) Nature 501:373,; McKracken (2014) Nature 516:400; Wang (2014) Nat Medicine 20:616, Mark Scott (unpublished)
  5. 5. Ways to make hPSC generate specific cell types or phenotypes 5 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. 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. 6 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. 7. Currently using high throughput method for generating cell types using complete set of human TFs 7 • 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. 8. Rapid generation of neurons, myocytes, and endothelium using TFs identified by TFome screen 8 4 day TF induction
  9. 9. Ways of engineering tissues and organoids 9 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. 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 10 necrotic core Lancaster (2013) Nature 501:373
  11. 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. 11 Kolesky (2104) Adv Mater 26:3124
  12. 12. We are getting close to generating cerebrovascular organoids 12 Organoid exhibiting layers of neural types and endothelial cells Lumen formation in hiPSC-derived endothelium Images courtesy of Mark Scott
  13. 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. 14. Ethical implications 14
  15. 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 15
  16. 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† 16 * Denker (2014) Cells Tissues Organs 199:221; Pera (2015) Nat Methods 12:917; Hyun (2016) Nature 533:169 † ISSCR (2016) Guidelines for Stem Cell Science and Clinical Translation, sections,, Glossary entry Embryo-like structures Warmflash (2014) Nat Methods 11:847
  17. 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 17 Images: , 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. 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. 18 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. 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 19 recognize Result: Current guidelines … don’t recognize
  20. 20. Research into human development involving embryos and SHELEs: Contrasting concepts of ethical guidelines 20 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. 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 … 21 • Ideally we should aim for integrated guidelines for embryos, hPSC, and SHELEs
  22. 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. 22
  23. 23. Thank you 23
  24. 24. Extra slides 24
  25. 25. Nociception and Pain development 25 Lee (2005) JAMA 294:297
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