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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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

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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Key directions in human embryo and pluripotent stem cell research

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hPSC

Direct differentiation to diverse cell types

DAPI

DAPI

DAPI

neur

on

mus

cle

endo

thel

ial

Develop organoids from embryoid bodies

cere

bral

ga

stric

many others…

• https://embryology.med.unsw.edu.au/embryology/images/9/98/Human_embryo_day_5.jpg

• Gist Croft, Cecilia Pelligrini, Ali H. Brivanlou, Rockefeller University

Human embryos

5d blastocyst

12d embryo

Engineered hPSC-based tissues & organoids

card

iom

yocy

tes

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 (unpublished); Lancaster et.al.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)

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

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

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

Rapid generation of neurons, myocytes, and endothelium using TFs identified by TFome screen

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4 day TF induction

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

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

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

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

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

13 Images courtesy of Mark Scott

Ethical implications

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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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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

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

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

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

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

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

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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Thank you

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Extra slides

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Nociception and Pain development

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Lee (2005) JAMA 294:297

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