As described in the President's Council on Bioethics' recent White Paper, altered nuclear transfer (ANT) is a broad conceptual proposal for producing pluripotent stem cells without creating and destroying embryos. In the description set forth below, we outline a research program for a form of ANT that should allow us to produce pluripotent stem cells without creating or destroying human embryos and without producing an entity that undergoes or mimics embryonic development. The method of alteration here proposed (oocyte assisted reprogramming) would immediately produce a cell with positive characteristics and a type of organization that from the beginning would be clearly and unambiguously distinct from, and incompatible with, those of an embryo. Incapable of being or becoming an embryo, the cell produced would itself be a pluripotent cell that could be cultured to establish a pluripotent stem cell line. Significantly, this cell would not be totipotent, as a zygote is.

Our proposal is for initial research using only nonhuman animal cells. If, but only if, such research establishes beyond a reasonable doubt that oocyte assisted reprogramming can reliably be used to produce pluripotent stem cells without creating embryos, would we support research on human cells.

With few exceptions all human cells contain a complete human genome, i.e. the complete DNA sequence characteristic of the human species. Specifically, one-celled human embryos, pluripotent human embryonic stem (or ES) cells, multipotent human adult stem cells, and differentiated (specialized) adult human cells such as neurons all contain a complete human genome. Thus, possession of a human genome is a necessary but not sufficient condition for defining a human embryo with its inherent dignity. Rather the nature of each cell depends on its epigenetic state, i.e. which subset of the approximately thirty thousand human genes is switched on or off and, if on, at what level. For example, the gene for albumin, a liver specific protein, is found both in human embryos and in adult human liver cells called hepatocytes. However, neither the messenger RNA (mRNA) for albumin nor the protein itself is found in single-celled embryos because in them the gene is silenced.

This fundamental observation has given rise to the concepts of cell fate plasticity and epigenetic "reprogramming." If successful, reprogramming converts a cell from one kind to another by changing its epigenetic state. The ability to clone animals, such as Dolly the sheep, by transfer of a specialized adult nucleus to an enucleated oocyte demonstrates the power of epigenetic reprogramming: the oocyte cytoplasm is sufficient to reprogram the somatic nucleus to a totipotent state. Human cloning has been proposed as a means of generating human embryos whose pluripotent stem cells would be used in scientific and medical research. Here, through a form of altered nuclear transfer, we propose to utilize the power of epigenetic reprogramming in combination with controlled alterations in gene expression to directly produce pluripotent cells using adult somatic nuclei, without generating and subsequently destroying embryos.

How do pluripotent stem cells differ from totipotent single-celled embryos? Several key transcription factors essential for establishing and maintaining the pluripotent behavior of ES cells have been identified. Importantly, some of these are specifically expressed only in pluripotent cells, such as embryonic stem cells or the cells found in the inner-cell-mass (ICM) of the week-old embryo or blastocyst. They are not expressed in oocytes or single-celled embryos. Expression of these factors therefore positively defines and distinguishes mere pluripotent cells from embryos. These factors instruct a cell to have the identity of a pluripotent cell. Currently, the best studied example is the homeodomain transcription factor called nanog (Mitsui, Tokuzawa et al. 2003*). Nanog is not present in oocytes or single-celled embryos, but first becomes expressed weakly in the morula and then highly in the ICM (Mitsui, Tokuzawa et al. 2003; Hatano, Tada et al. 2005). Deletion of nanog does not prevent early cleavage stages of embryogenesis including formation of the ICM but does prevent the formation of an epiblast (Mitsui, Tokuzawa et al. 2003). ES cells in which nanog is blocked lose their pluripotency -- which clearly shows that nanog is a positive factor instructing cells to be pluripotent, i.e. to behave like an ES cell. Furthermore, ES cells which constitutively express nanog can no longer be differentiated, i.e. are forced to remain in their undifferentiated state (Mitsui, Tokuzawa et al. 2003).

We propose a procedure that combines epigenetic reprogramming of a somatic nucleus with forced expression of transcription factors characteristic of embryonic stem cells, to produce a pluripotent stem cell. As a result of this procedure, nanog and/or other, similar factors,¹ would be expressed at high levels in somatic cells prior to nuclear transfer, to bias the somatic nucleus towards a pluripotent stem cell state. Such altered nuclei would then be epigenetically reprogrammed by transplantation into enucleated oocytes. Alternatively or concomitantly, the mRNA for these same factors could be introduced into the oocyte prior to nuclear transfer. This procedure could ensure that the epigenetic state of the resulting single cell would immediately be different from that of an embryo and like that of a pluripotent stem cell: the somatic-cell nucleus would be formed into a pluripotent stem-cell nucleus and never pass through an embryonic stage. Therefore, unlike some other proposed methods of ANT, this method would achieve its objective not by a gene deletion that precludes embryonic organization in the cell produced, but rather by a positive transformation that generates, ab initio, a cell with the distinctive molecular characteristics and developmental behavior of a pluripotent cell, not a totipotent embryo. This should allow us to produce a pluripotent stem cell line with controlled genetic characteristics.

ENDORSERS

Institutional affiliations are provided for purposes of identification only and do not necessarily represent the views of organizations with which endorsers are affiliated. Endorsers who are not themselves specialists in biomedical science do not put themselves forward as experts in that field. Their endorsement of the proposal pertains to the ethics of ANT- OAR, assuming its technical feasibility.

Hadley Arkes, Ph.D.
Edward N. Ney Profesor of Jurisprudence and American Institutions
Amherst College
Amherst, Massachusetts

Rev. Nicanor Pier Giorgio Austriaco, O.P., Ph.D.
Assistant Professor of Biology
Providence College
Providence, Rhode Island

Rev. Thomas Berg, L.C., Ph.D.
Executive Director
The Westchester Institute for Ethics and the Human Person
Thornwood, New York

E. Christian Brugger, D. Phil.
Assistant Professor of Theology
Institute for Psychological Sciences
Arlington, Virginia

Nigel M. de S. Cameron, Ph.D.
President, Institute on Biotechnology and the Human Future
Research Professor of Bioethics
Chicago-Kent College of Law, Illinois Institute of Technology
Chicago, Illinois

Joseph Capizzi, Ph.D.
Catholic University of America
Fellow, Culture of Life Foundation
Washington, D.C.

Maureen L. Condic, Ph.D.
Associate Professor of Neurobiology
University of Utah, School of Medicine
Salt Lake City, Utah

Samuel B. Condic, M.A.
Department of Social Sciences
University of Houston - Downtown
Houston, Texas

Rev. Kevin T. FitzGerald, S.J., Ph.D.
Dr. David P. Lauler Chair in Catholic Health Care Ethics
Center for Clinical Bioethics Research
Associate Professor Department of Oncology
Georgetown University Medical Center
Washington, D.C.

Rev. Kevin Flannery, S.J., D.Phil.
Dean of the Philosophy Faculty
Pontifical Gregorian University
Rome, Italy

Edward J. Furton, Ph.D.
Ethicist
The National Catholic Bioethics Center
Philadelphia, Pennsylvania

Robert P. George, J.D., D.Phil.
McCormick Professor of Jurisprudence
Princeton University
Princeton, New Jersey

Timothy George, Th.D.
Dean
Beeson Divinity School
Samford University
Birmingham, Alabama

Alfonso Gómez-Lobo, Dr. phil.
Ryan Professor of Metaphysics and Moral Philosophy
Georgetown University
Washington, D.C.

Germain Grisez, Ph.D.
Flynn Professor of Christian Ethics
Mount Saint Mary's University
Emmitsburg, Maryland

Markus Grompe, M.D.
Director
Oregon Stem Cell Center
Portland, Oregon

John M. Haas, Ph.D.
President
The National Catholic Bioethics Center
Philadelphia, Pennsylvania

Robert Hamerton-Kelly, Th.D.
Dean of the Chapel (Retired)
Stanford University
Palo Alto, California

John Collins Harvey, M.D., Ph.D.
Senior Research Scholar and Professor Emeritus of Medicine
Center for Clinical Bioethics
Georgetown University Medical Center
Washington, D.C.

Paul J. Hoehner, M.D., M.A., FAHA
Harvey Fellow in Theology, Ethics and Culture, The University of Virginia Graduate School of Arts and Sciences
Associate Professor of Anesthesiology,
The University of Virginia Health Sciences Center
Charlottesville, Virginia

William B. Hurlbut, M.D.
Consulting Professor in the Program in Human Biology
Stanford University
Palo Alto, California

John F. Kilner, Ph.D.
President
The Center for Bioethics and Human Dignity
2065 Half Day Road
Bannockburn, Illinois

Patrick Lee, Ph.D.
Professor of Philosophy
Franciscan University of Steubenville
Steubenville, Ohio

William E. May, Ph.D.
Michael J. McGivney Professor of Moral Theology
John Paul II Institute for Studies on Marriage and Family at
The Catholic University of America
Washington, D.C.

Rev. Gonzalo Miranda, L.C., Ph.L., S.T.D.
Dean of Bioethics
Regina Apostolorum Pontifical Athenaeum
Rome, Italy

C. Ben Mitchell, Ph.D.
Associate Professor of Bioethics & Contemporary Culture
Trinity International University
Bannockburn, Illinois

Most Reverend John J. Myers, J.C.D., D.D.
Roman Catholic Archbishop of Newark, New Jersey

Chris Oleson, Ph.D.
Associate Professor of Philosophy
Center for Higher Studies
Thornwood, New York

Rev. Tad Pacholczyk, Ph.D.
Director of Education
The National Catholic Bioethics Center
Philadelphia, Pennsylvania

Rev. Peter F. Ryan, S.J., S.T.D.
Associate Professor of Moral Theology
Mount St. Mary's University
Emmitsburg, Maryland

William L. Saunders, J.D.
Senior Fellow and Director
The Center for Human Life & Bioethics
The Family Research Council
Washington, D.C.

David Stevens, M.D., M.A.
Executive Director
Christian Medical & Dental Associations
Bristol, Tennessee

Rev. Msgr. Stuart W. Swetland, S.T.D.
Director
The Newman Foundation
Adjunct Associate Professor
University of Illinois at Urbana-Champaign
Urbana, Illinois

M. Edward Whelan III, J.D.
President
Ethics and Public Policy Center
Washington, D.C.

Rev. Thomas Williams, L.C., Ph.L., S.T.D.
Dean of Theology
Regina Apostolorum Pontifical Athenaeum
Rome, Italy

NOTES

1. Nanog is only one example of a growing list of candidate factors, numbering probably at least 10. Oct3/4 is another well-studied example (3) and is noteworthy because it is also expressed at high levels in pluripotent adult stem cells.

REFERENCES CITED (WITH ABSTRACTS)

Hatano, S. Y., M. Tada, et al. (2005). "Pluripotential competence of cells associated with Nanog activity." Mech Dev 122(1): 67-79.

Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.

Mitsui, K., Y. Tokuzawa, et al. (2003). "The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells." Cell 113(5): 631-42.

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.

ADDENDUM

At a recent gathering of many of the original signers of this statement, we reaffirmed our support for continued scientific exploration of ANT/OAR in animal models in the following terms:

In light of recent research further delineating the very early processes that control the transition from totipotency to pluripotency during mammalian embryonic development*, we are in agreement that immediate, constructive, and ethically acceptable research (using animal models) can be pursued that will significantly clarify the prospects of obtaining cells with properties equivalent to those of human pluripotent stem cells.  This research will be within the parameters already outlined in earlier proposals for ANT/OAR research, specifically focusing on the strong determination of the pluripotent state for a nuclear-transfer derived cell by means of transcription factor regulation/overexpression, including the possibility that functional deletion of developmental genes like cdx-2 in the somatic nucleus may be able to assist in modifying regulatory loops for these factors. By limiting and carefully constraining the reprogramming power of the oocyte through these approaches, such that a totipotent cell is precluded as a reprogramming outcome, it may be possible to directly derive a cell that is the functional equivalent of a pluripotent stem cell. Considering the scientific, medical, political, and social benefits to be gained by this research, we recommend that it be supported and pursued by all those desirous of the advances of human embryonic stem cell research without the creation or destruction of human embryos.

[* See for instance, Deb K, Sivaguru M, Yong HY, Roberts RM. “Cdx2 gene expression and trophectoderm lineage specification in mouse embryos.”  Science 2006 Feb 17;311(5763):992-6;  Niwa H, Toyooka Y, Shimosato D, Strumpf D, Takahashi K, Yagi R, Rossant J., “Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation.” Cell. 2005 Dec 2;123(5):917-29.]