Stem Cell Research and Cloning N

Doing Science in a

Pluralistic Society

David Lemberg, M.S., D.C. Associate Faculty National University 1


“There are many different ends that men may seek and still be fully rational, fully men, capable of understanding each other and sympathizing and deriving light from each other, . . . worlds, outlooks, very remote from our own.”

— Isaiah Berlin, The Pursuit of the Ideal

“Yet with how many things are we upon the brink of becoming acquainted, if cowardice or carelessness did not restrain our inquiries.”

— Mary Shelley, Frankenstein

“Questions of use of science and technology are always moral and political questions, never simply technical ones.”

— Leon Kass, The New Biology: What Price Relieving Man’s Estate?


I’m not a mistake,

I’m not a fake,

It’s set in my DNA

—Miley Cyrus

“Can’t Be Tamed”





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Birth of Dolly —announced February

1997

Cloning of humans was universally deemed imminent

“The cloning of humans would fit precisely into Adolph Hitler’s world view”

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—Die Welt (2-27-97)

Cloning of human beings would permit

“a eugenic and racist selection of the human race”

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—European Parliament (3-13-97)


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In 1903 plant physiologist Herbert John

Webber described clons

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Groups of plants that are propagated by the use of any form of vegetative parts such as bulbs, tubers, cuttings, . . . which are simply parts of the same individual”

Clon(e) is derived from the Greek klon meaning “trunk” or “branch”


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In 1994 cloning was described as “taking the nucleus of a cell from the body of an adult and transferring it to an unfertilized egg, destroying the genome of the oocyte of the egg, and letting it develop”*

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Two years before the birth of Dolly

*Voelker R: A clone by any other name is still an ethical concern. JAMA 271:331–332, 1994


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In 1894 Jacques

Loeb cleaved fertilized sea urchin eggs

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In some experiments two entire embryos would develop from the original zygotic material


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In 1914 Hans

Spemann pinched off a portion of cytoplasm in salamander zygotes

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If a nucleus migrated to the cytoplasm from one of the blastocyst cells, two embryos would result


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The origins of SCNT date from the 1950s and

1960s

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Experiments on leopard frogs (Rana pipiens)

In a notable understatement, Robert

Briggs and Thomas King declared “although the method of nuclear transplantation should be valuable principally for the study of nuclear differentiation, it may also have other uses ”



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The next stage would be to clone mammals

1983 — James McGrath and Davor Solter of the Wistar Institute in Philadelphia utilized SCNT to clone mice

In 1993 four calves were cloned from cultured cells derived from the inner cell mass of blastocysts


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Dolly’s birth was a big breakthrough — cloning a mammal from an adult somatic cell

Before Dolly, in October 1993, Jerry Hall created cloned human embryos by fission of a single human embryo

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This method was a common technique in animal husbandry



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Robert Stillman, the director of the IVF program at George Washington

University said the purpose was to help infertile couples who have “just the basic human desire to have a family”

This is exactly a primary justification to proceed with research into human cloning


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Very interestingly, Robert McKinnell at the University of Minnesota stated “it was much harder to take a cell from an adult organism and use it to make embryos or even clones, since the cells of adult organisms are committed to specific functions and have switched off their capacity for full development”

Both of these hurdles have now been stunningly overcome


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The outcry against Hall’s work was immediate

George Annas—bioethicist

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“This is the experiment we were never going to do— it’s a horror story”

Others suggested the public is

“uncomfortable with meddling with the life-reproducing process”


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The Vatican’s official newspaper,

L’Osservatore Romano, stated the U.S. must regulate “unscrupulous scientists who venture into a tunnel of madness”

In contrast, medical ethicist Norman Fost said these decisions should be the parents’ prerogative — “a presumption of privacy and liberty, that people should be able to live their lives the way they want and to make babies the way they want”


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Birth of Dolly —announced February

1997

Cloning of humans was universally deemed imminent

“The cloning of humans would fit precisely into Adolph Hitler’s world view”



—Die Welt (2-27-97)

Cloning of human beings would permit

“a eugenic and racist selection of the human race”

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—European Parliament (3-13-97)



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Wilmut’s team utilized SCNT

Dolly derived from a clone of an adult somatic cell

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These cell populations were clones of mammary gland cells of a 6-year-old ewe


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Remarkably the report of the isolation and growth of human stem cells soon followed the announcement of the birth of Dolly

James Thomson’s team at the University of Wisconsin cultured 5 independent cell lines from the ICMs of 14 blastocysts


Thomson Lab Stem Cells

Fig. 1. Derivation of the

H9 cell line. (A) Inner cell mass-derived cells attached to mouse embryonic fibroblast feeder layer after 8 days of culture, 24 hours before first dissociation.

Scale bar, 100 mm. (B)

H9 colony. Scale bar,

100 mm. (C) H9 cells.

Scale bar, 50 mm. (D)

Differentiated H9 cells, cultured for 5 days in the absence of mouse embryonic fibroblasts, but in the presence of human LIF (20 ng/ml;

Sigma). Scale bar, 100 mm.

Science 282(5391):1145-1147, 1998

Human Leukemia Inhibitory Factor

(LIF) is a lymphoid factor that promotes long-term maintenance of embryonic stem cells by suppressing spontaneous differentiation.


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Thomson’s paper was titled “Embryonic

Stem Cell Lines Derived from Human

Blastocysts”

The article concluded “progress in developmental biology is now extremely rapid. Human ESCs will link this progress even more closely to the prevention and treatment of human disease.”



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In August 2001, the same month Time placed Thomson on its cover, President

Bush severely restricted federal funding for stem cell research

Bush declared “Research on embryonic stem cells raises profound ethical questions, because extracting the stem cells destroys the embryo and thus destroys its potential for life.”


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Until two years ago, stem cells were exclusively derived from an embryo

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From the inner cell mass of a blastocyst




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Isolating stem cells from the ICM kills the embryo

Many institutions and organizations believe this is equivalent to killing a born human being


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Richard Doerflinger

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Associate Director of the U.S. Conference of

Catholic Bishops' Secretariat of Pro-Life

Activities

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Doerflinger states that embryo research

“directly promotes the destruction of embryonic human life” and destruction of embryos represents “wrongful killing”

Doerflinger RM: The ethics of funding embryonic stem cell research.

A Catholic viewpoint. Kennedy Inst Ethics J 9(2):137-150, 1999

Stem cells are “lethally harvested from embryos”


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The Catholic church, as a monolithic entity, is opposed to all extracorporeal activities involving embryos.

The Vatican’s Instruction Dignitas Personae advises the faithful —

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“The fruit of human generation, from the first moment of its existence, that is to say, from the moment the zygote has formed, demands the unconditional respect that is morally due to the human being in his bodily and spiritual totality”

Vatican: Congregation for the Doctrine of the Faith. Instruction Dignitas

Personae on Certain Bioethical Questions, September 8, 2008


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Dignitas Personae

Experimentation on human embryos

“constitutes a crime against their dignity as human beings . . . [and] always constitutes a grave moral disorder”

“The use of embryonic stem cells . . . even when these are provided by other researchers through the destruction of embryos . . . presents serious problems from the standpoint of cooperation in evil and scandal”


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On the previous views, the embryo is a human being with full moral status

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Embryo have all the rights and privileges that born human beings enjoy

The opposite view holds that the embryo has no moral status whatsoever

A middle ground may be sought in which the embryo holds no moral status but yet is entitled to moral respect


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As moral agents we acknowledge the moral value of embryos and accord them respect

We do embryo research to fulfill specific, meaningful, high-impact goals

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Improving assisted reproductive technologies

Improving contraception

Finding solutions to genetic diseases

But of course many remain vigorously opposed to such research


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Creating embryos specifically for research

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This is highly contentious

In his August 2001 Presidential Statement,

President Bush described the “deeply troubling” activity of creating embryos “solely to experiment on them”


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Instrumentalism — created human embryos are intended to be used as a

means only — the ends of the embryos are not considered

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Immanuel Kant’s Formula of Humanity as an End in Itself — “So act that you use humanity . . . always at the same time as an end, never merely as a means”

Wood AW: Kant. Oxford, UK, Blackwell, 2005, p 135

But — embryos are not sentient, they have no interests, and they have no ends


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Use of “spare” embryos

Spare embryos are produced in hyperovulation cycles and unused in

IVF procedures

Spare embryos are contrasted with embryos created specifically for research

Thus, the spare–created distinction


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In IVF, spare embryos are either frozen for later use or destroyed

Using these embryos for research represents a third alternative

But spare embryos are often not of top quality

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Many have various defects that caused them to be eliminated from the pool of candidates for implantation

Others have been stored for many years


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Research based on these [spare] embryos can lead to seriously misleading conclusions*

*Green RM: The Human Embryo Research Debates. New York, Oxford

University Press, 2001, p 15


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How to make federal choices in a pluralistic society?

Conflicts between personal autonomy and compelling state interests

Need for long overdue system of ethical review and oversight

Need for careful study, deliberation, and guidelines for the conduct of both research and clinical practice


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“Trend toward transplants in which the stem cells are expected to behave in different ways, even though there is little empirical evidence to suggest they can do so”

Report in June 2010 of the “discovery of strange lumps of cells in the kidney of a woman who had undergone stem-cell treatment in Thailand”

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Nature 465:997, 2010


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In Russia, a boy with ataxia telangiectasia (Louis-Bar syndrome; degeneration of the cerebellum, with dyscoordinated movement and speech) was injected with fetal neural stem cells

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Four years later he developed multifocal tumors in the brain stem and cauda equine

The tumors were derived from the injected fetal cells


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200 clinics worldwide – 100 in China alone – offering unproven SC treatments for scores of disorders including MS, ALS, and spinal cord injury

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Nature 459:147, 2009

The potential profits are huge

Few facilities offer evidence from controlled clinical trials or from rigorous follow-up of patients


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 iPSCs may substitute for embryonic stem cells

In 2007 two teams simultaneously announced the creation of iPSCs

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Yamanaka et al at Kyoto University*

Thomson et al at the University of

Wisconsin**

*Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676, 2006

**Yu J, et al: Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917-1920, 2007.


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The Yamanaka team utilized four transcription factors to reprogram adult differentiated cells to a pluripotent state

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OCT4, SOX2, KLF4, and c-MYC

This work was groundbreaking

The primary obstacle going forward was the use of a retroviral vector to deliver the transcription factors


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Importantly, the creation of iPSCs does not involve the destruction of embryos

The White House said that President

Bush was “very pleased” about the new findings, adding that “By avoiding techniques that destroy life, while vigorously supporting alternative approaches, President Bush is encouraging scientific advancement within ethical boundaries.”


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Great strides have been made in a very short time iPSCs can now be generated using a multiprotein expression vector which is readily integrated into the host cell genome

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The entire construct can easily be removed using a transposase enzyme


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 iPSCs have been successfully used for therapy in animal models

The genetic defect for sickle cell

anemia has been corrected in mice*

*Hanna J, et al: Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318(5858):1920–1923, 2007.


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 iPSCs were generated by reprogramming somatic cells from sickle cell mice

Genetic defect was corrected using standard recombination techniques

The reengineered iPSCs were caused to differentiate to hematopoietic stem cells

These cells were transplanted into humanized sickle cell anemia mice


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Correction of a genetic deficiency responsible for

Duchenne muscular dystrophy (DMD) in a mouse model of the disease*

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A human artificial chromosome (HAC) was created, containing a complete genomic dystrophin sequence

The HAC was transferred into iPSCs derived from

DMD-mice

Differentiation of iPSCs into muscle stem cells

Transplantation of genetically corrected muscle cells into the DMD-mice

Restoration of dystrophin expression proved the principle of treatment utilizing gene therapy combined with iPSC transformation*

*Kazuki Y, et al: Complete genetic correction of iPS cells from Duchenne muscular dystrophy. Mol Ther 18(2):386–393, 2010.



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 iPSCs can be efficiently differentiated into neural precursor cells which could be used in treatment of a variety of neurologic disorders iPSC-derived neurons have been transplanted into the brains of adult rats modeling Parkinson’s disease

Functional recovery was demonstrated in eight of nine animals*

*Wernig M, et al: Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 105(15):5856-5861, 2008.


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Therapies based on iPSCs could provide assistance for tens of millions of people worldwide iPSCs may even lead to new treatments for infertility

15% of Americans are affected by infertility


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 iPSCs enables development of in vitro models to investigate the formation of various human reproductive cell types

Human iPSCs have already been used to generate primordial germ cells*

Breakthroughs could lead to the generation of functional sperm and eggs

Provide gametes that are genetically identical to a parent, eliminating the necessity of using an unrelated gamete donor

*Park TS et al: Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells. Stem Cells 27(4):783–795, 2009


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Create sperm and eggs from tissue of unexpectedly deceased spouses and partners

Create eggs from tissue of postmenopausal women

Create sperm from iPSCs of female origin and eggs from iPSCs of male origin


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Tetraploid blastocyst complementation has recently been used to produce adult fertile mice from iPSCs*

Proved that differentiated cells can be restored to an embryonic level of pluripotency with complete developmental potential

* Boland MJ, et al: Adult mice generated from induced pluripotent stem cells. Nature 461:91–95,

2009


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TBC was developed to characterize the full developmental potential of embryonic stem cells

Mouse tetraploid embryos are created by electrically fusing blastomeres in two-cell embryos

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Creating 4N cell


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Murine ESCs are aggregated with tetraploid embryos and the aggregates are transferred into the uterus of 2.5-day pseudopregant recipients

In TBC, all adult tissues derive from the stem cell line and extraembryonic tissues derive from the tetraploid cells


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The initial report by Andràs Nagy’s team described the successful creation of live murine offspring.*

The Boland team at Scripps team extended the method to create live fertile offspring utilizing iPSCs

*Nagy A, et al: Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc Natl Acad

Sci USA 90(18):8424–8428, 1993.


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Very short conceptual leap from cloning mice to cloning humans

Parties categorically opposed to cloning have recommended closing previously unrecognized loopholes in “current laws and policies, enacted before iPSCs were contemplated”


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The progress of science cannot be predicted and additional novel procedures with reproductive cloning potential are likely to be developed

All scientific experimentation is potentially world-changing

New scientific developments often have novel and unanticipated applications


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“When legitimate uses of a technique are likely, regulatory policy should avoid prohibition and focus on ensuring that the technique is used responsibly for the good of those directly involved”*

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“In the long run, our hope can only lie in education: in a public educated about the meanings and limits of science and enlightened in its use of technology”**

*Robertson J: Human cloning and the challenge of regulation. NEJM 339(2):119–122, 1998

**Kass LR: The new biology. What price relieving man’s estate? Science 174(4011):779–788, 1971.


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“Is there knowledge which should not be pursued because it is noxious to life?”

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—Friedrich

Nietzsche (1844–

1900)


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“Knowledge presupposes life and so has the same interest in the preservation of life which every being has in its own continuing existence. Thus science requires a higher supervision and guarding: a hygiene of life is placed close beside science.”

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—Nietzsche, On the Advantage and

Disadvantage of History for Life


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Need to do research on humans

Unsanctioned research could proceed in the presence of a ban or the absence of regulatory guidelines

More than 100,000 IVF procedures are now done annually in the U.S.


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“Sometime pretty soon a child conceived through cloning will appear on the front pages of all the newspapers in the world.

. . . And within 30 days there will be a cloning clinic in California.”*

The fertility industry is almost completely unregulated

* Eibert MD: Human cloning. Myths, medical benefits, and constitutional rights. Hastings Law J 53(5):1097–

1116, 2002


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SC research and the prospect of human cloning —latest battleground centered on

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The complex interrelationships between humankind and nature

Decision-making in a pluralistic society

Procreative liberty

Conflicts between personal autonomy and compelling state interests


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Our collective stance on stem cell research and the closely related topic of reproductive cloning

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A proxy not only for how we view the giveand-take between science and society, but also for who and what we, as a society, are choosing to be


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Are we, in fact, a pluralistic society or a polarized collation of individuals?

Are we stakeholders or are we placeholders?


Stem Cell Research and Cloning N

Doing Science in a

Pluralistic Society

Until two years ago, stem cells were exclusively derived from an embryo

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Stem Cell Research and Cloning N

Doing Science in a

Pluralistic Society

Until two years ago, stem cells were exclusively derived from an embryo

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