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OCR for page 108
Colloquium
Therapeutic cloning in the mouse
Peter Mombaerts*
The Rockefeller University, 1230 York Avenue, New York, NY 10021
Nuclear transfer technology can be applied to produce autologous
differentiated cells for therapeutic purposes, a concept termed
therapeutic cloning. Countless articles have been published on the
ethics and politics of human therapeutic cloning, reflecting the
high expectations from this new opportunity for rejuvenation of
the aging or diseased body. Yet the research literature on thera-
peutic cloning, strictly speaking, is comprised of only four articles,
all in the mouse. The efficiency of derivation of embryonic stem cell
lines via nuclear transfer is remarkably consistent among these
reports. However, the efficiency is so low that, in its present form,
the concept is unlikely to become widespread in clinical practice.
Cloning of mammals by nuclear transfer was introduced in
1986 (1) and gained attention by the report of the birth of
Dolly the sheep in 1997 (24. To date, seven mammalian species
have been cloned from adult cells (3), with the notable exception
of primates (4~. Mouse cloning has been possible since 1998 (5~.
The versatility of this small mammal has propelled the mouse as
the experimental model of choice for developing new cloning
strategies and applications.
Therapeutic cloning, in its current embodiment, entails the
derivation of embryonic stem (ES) cell lines from an already
born organism (64. This individual may suffer from a disease that
is potentially responsive to autologous cell replacement. ES cell
precursors appear transiently at the blastocyst stage in early
embryonic development, and their cultured ES cell derivatives
have the unrivaled ability to differentiate, in vitro and in viva, into
any specialized cell type. How can cells with ES properties then
be derived from a fully grown organism that no longer contains
ES cells? Nuclei isolated from a tissue biopsy of an adult
organism can be transferred into oocytes. Some of these recon-
structed embryos develop into blastocysts, from which ES cell
lines can be derived. Provisionally, these cell lines are considered
to harbor the same special and highly desirable properties as
"conventional" ES cell lines derived from normal embryos
produced by fertilization; but to distinguish them from such lines,
they are referred to as nuclear transfer ES (ntES) cell lines. The
ntES cell lines are expected to be genetically identical to the
nucleus donor (the fully grown organism) except for the mito-
chondrial genome, which is derived from the oocyte. This
near-genetic identity holds the promise of circumventing im-
mune rejection of cells originating by differentiation from ES
cells, derived from a random embryo that will differ genetically
from the diseased individual. Although rejection of foreign cells
can be prevented or contained, it remains the bane of trans-
plantation medicine.
An attractive scenario has thus emerged in which each of us
could have a few ntES cell lines derived while we are young and
healthy, perhaps even shortly after birth. When a disease arises
that can be treated by cell replacement, or when our aging body
is wearing out due to cell loss, our personal ntES cells would be
thawed out, expanded, and differentiated into the desired cell
type. Cells would be purified and transplanted to restore the
compromised cell function. Various differentiated cell types
could be combined to form tissue, organ parts or entire organs,
replacing, for instance, a defective heart valve.
11924-11925 1 PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1
Mouse ntES Cell Lines
The first report of a mouse ntES cell line appeared in 2000.
Munsie et al. (7) used cumulus cells, somatic cells that surround
the oocyte when it ovulates in the female, to generate 10
blastocysts, from which a single ntES cell line was developed.
This cell line was able to differentiate in vitro and in vivo, and
generated chimeric mice when injected into blastocysts. Shortly
thereafter, a total of five ntES-cell like lines were reported,
derived from 101 blastocysts produced from nuclei of neural
cells. Because the donor cells were of fetal origin, this paper does
not fall within the concept of therapeutic cloning, in its strict
sense (84. A third study appeared in 2001 and reported the
generation of 35 ntES cell lines from 398 blastocysts, generated
both from cumulus cells and tail tip cells (94. The distinct
advantages of using tail tip cells are that males also have tails,
and that a tail biopsy is relatively noninvasive compared with the
isolation of cumulus cells; a tail tip biopsy can be regarded as the
mouse equivalent of a human skin biopsy. The mouse ntES cells
could be induced to differentiate massively into dopaminergic
neurons, the cell type lost in Parkinson's disease in human.
The final two articles appeared in 2002 (10, 11~. Two ntES cell
lines were produced from 41 blastocysts generated by nuclear
transfer from B and T lymphocytes of peripheral lymph nodes,
demonstrating that the genome of these terminally differenti-
ated cells cart be reprogrammed (10~. The culmination of the
history of therapeutic cloning is a report of the first "therapeu-
tic" effect in the final study (114: one ntES cell line was generated
from 27 blastocysts generated by nuclear transfer from tail tip
cells of a profoundly immunodeficient mouse, homozygous for a
knockout mutation in the recombination activating gene 2. The
knockout mutation was "cured" by gene targeting, and the
manipulated ntES cells were differentiated in vitro into hema-
topoietic cells, which partially repopulated the crippled immune
system of other knockout mice with the same mutation. This is
a proof of principle of "therapy," although the experimental
design was somewhat artificial.
A high priority is now to demonstrate significant therapeutic
benefit of ntES cell derivatives in a model of a natural disease
such as type 1 diabetes. Moreover, differentiated cells derived
from a ntES cell line established from a sick mouse must be
transplanted into the same individual, not in another mouse of
the same inbred strain.
Efficiency
These reports make use of the same nuclear transfer technique:
piezo-actuated microinjection of somatic cell nuclei into enu-
cleated oocytes. The efficiency, or perhaps better, the lack
thereof, is remarkably consistent (Table 1~. Not all data are
This paper results from the Arthur M. Sackier Colioquium of the National Academy of
Sciences, "Regenerative Medicine," held October 18-22, 2002, at the Arnold and Mabel
Beckman Center of the National Academies of Science and Engineering in Irvine, CA.
Abbreviations: ES, embryonic stem; ntES, nuclear transfer ES.
*E-maii: peter@rockefeiler.edu.
(if) 2003 by The National Academy of Sciences of the USA
www.pnas.org/cgi/doi/10. 1 073/pnas. 1934141100
OCR for page 109
Table 1. Efficiency of n+ES cell derivation
Ref.
7
9
10
1 1
Tota I
Reconstructed
oocytes Blastocysts
362
1,016
980
202
2,560
10
398
41
27
476
ntES cell
lines
35
2
1
39
Efficiency, %
Per Per
oocyte blastocyst
0.3
3.4
0.2
0.5
0.15
available for each experiment, and some groups report the
number of successfully reconstructed oocytes but not the total
number of oocytes used for nuclear transfer. With these limita-
tions in mind, 4-16% of cloned blastocysts generated an ntES
cell line. Overall the efficiency is 8.2%: l ntES cell line can be
derived from 12 cloned blastocysts. Unfortunately, none of these
studies contains control data for ES cell lines derived in parallel
from blastocysts produced from fertilized oocytes, but this value
is expected to be >8.2%. The number of oocytes required to
generate one ntES cell line in these reports, a number that is
critical for human applications, as argued below, varies from as
high as ~l,000 to as low as ~15 (for cumulus cells from B6D2Fl
females; ref. 9~. In the most comprehensive study, performed by
a single experimentator over a short period, ~30 reconstructed
oocytes were needed to produce one ntES cell line (9~.
The testing of the ntES cell lines is incomplete in these reports,
and germ-line transmission, the gold standard for pluripotency
of an ES cell line, has been demonstrated only for a few lines and
not in all reports. A cautious objective for clinical applications is
to produce two to three ntES cell lines per individual, realizing
that some of these may be aneuploid or not sufficiently pluri-
potent. This requires, in the best case and in the best hands,
60-90 successfully reconstructed oocytes, say, 100 oocytes avail-
able for micromanipulation.
Extrapolation to Humans
This analysis of the limited body of literature raises concerns
about the feasibility and relevance of therapeutic cloning, in its
1. Willadsen, S. M. (1986) Nature 320, 63-65.
2. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. & Campbell, K. H. S. (1997)
Nature 385, 810-813.
3. Wilmut, I., Beaujean, N., de Sousa, P. A., Dinnyes, A., King, T. J., Paterson,
L. A., Wells, D. N. & Young, L. E. (2002) Nature 419, 583-586.
4. National Academy of Sciences (2002) Scientific and Medical Aspects of Ht''na'
Rep~odt~ctive Cloning (Natl. Acad. Press, Washington, DC).
5. Wakayama, T., Perry, A. C. F., Zuccotti, M., Johnson, K. R. & Yanagimachi,
R. (1998) Nat~'re 394, 369-374.
6. Hochedlinger, K. & Jaenisch, R. (2003) N. Ethel. J. Med. 349, 275-286.
7. Munsie, M. J., Michalska, A. E., O'Brien, C. M., Trounson, A. O., Pera,
Mombaerts
current embodiment, for human clinical practice. Nuclear trans-
fer is unlikely to be much more efficient in human than in mouse.
Optimistically, ~l00 human oocytes would be required to gen-
erate customized ntES cell lines for a single individual. A crucial
difference is that, although 100 mouse oocytes can be obtained
0 from a few superovulated females at a cost of ~$20, human
8 8 oocytes must be harvested from superovulated volunteers, who
5 are reimbursed for their participation. Add to this the complexity
4 of the clinical procedure, and the cost of a human oocyte is
~$1,000-2,000 in the U.S. Thus, to generate a set of customized
8.2 ntES cell lines for an individual, the budget for the human oocyte
material alone would be ~$100,000-200,000. This is a prohibi-
tively high sum that will impede the widespread application of
this technology in its present form.
Despite major efforts, the efficiency of nuclear transfer has
not increased over the years in any of the mammalian species
cloned. Little hope should be placed in a dramatic (say, l0-fold)
increase in efficiency in the near future. It becomes imperative
to develop alternative strategies for therapeutic cloning, if this
approach is ever to make a significant impact on medicine.
Alternative strategies can be divided into oocyte-dependent
and oocyte-independent approaches. First, oocytes could be
differentiated from existing ES cell lines, so that they can be
produced in essentially unlimited numbers. This would eliminate
completely the need for human oocyte donors. This exciting new
approach has become realistic with a recent report of oocytes
derived from mouse ES cells (124. For therapeutic cloning
purposes, the oocyte is essentially a processor for reprogram-
ming the inserted nucleus, and its nuclear genome is not carried
over in the ntES cells. Another strategy would be to use oocytes
from another species, ideally a nonprimate species such as rabbit.
However, the idea of generating embryos with mixed human/
animal properties, even transiently, is offensive to many people.
In the long run, efforts should be concentrated toward devel-
oping oocyte-independent systems, for instance by fusing so-
matic cells with enucleated ES cells, or by injecting ES cell- or
oocyte-derived reprogramming factors into somatic cells. A
major benefit of the complete elimination of oocytes and
embryos from the concept of therapeutic cloning is that the
ethical debate would vaporize instantaneously. In this way,
scientific progress may provide a solution to ethical concerns.
10.
M. F. & Mountford, P. S. (2000) Curry: Biol. lo, 989-992.
8. Kawase, E., Yamazaki, Y., Yagi, T., Yanagimachi, R. & Pedersen, R. A. (2000)
Genesis 28, 156-163.
9. Wakayama, T., Tabar, V., Rodriguez, I., Perry, A. C. F., Studer, L. &
Mombaerts, P. (2001) Science 292, 740-743.
i. Hochedlinger, K. & Jaenisch, R. (2()()2) Natr''e 415, 1035-1038.
11. Rideout, W. M., III, Hochedlinger, K., Kyba, M., Daley, G. Q. & Jaenisch, R.
(20()2) Cell 109, 17-27.
12. Huhner, K., Fuhrmann, G., Christenson, L. K., Kehler, J., Reinhold, R., De La
Fuente, R., Wood, J., Strauss, J. F., Ill, Boiani, M. & Scholer, H. R. (2003)
Science 300, 1251-1256.
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11925
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OCR for page 111
~-
-'
-
c_ Arthur M. Sack/er
C O L L O Q U I A
OF THE NATIONAL ACADEMY OF SCIENCES
Regenerative Medicine
October 18-22, 2002
Arnold and Mabel Beckman Center, Irvine, CA
Organized by Inder M. Verma and Fred H. Gage
Program
Friday, October 18
Keynote Speaker
John Gurdon, Wellcome CRC Institute, University of Cambridge
Stem Cells
Saturday, October 19
Stem Cell Biology I: Species
Chair, Susan Bryant
Jennifer Fletcher, University of California, Berkeley
Stem Cell Populations in Plants
Elaine Fuchs, The Rockefeller University
Beauty Is Skin Deep: Skin Stem Cells and Their Lineages*
Austin Smith, University of Edinburgh
Pluripotency and Lineage Determination of Mouse Embryonic Stem Cells
Irving Weissman, Stanford University School of Medicine
Biology of Murine Stem and Progenitor Cells
*Lounsbury Lecture
Stem Cell Biology 11: Systems
Chair, Inder M. Verma
Michael German, University of California, San Francisco
Differentiation of Pancreatic Beta-Cells from Progenitor Cells
Catherine M. Verfaille, University of Minnesota Medical School
Unexpected Potential of Adult Stem Cells
Phil Newmark, University of Illinois Urbana-Champaign
Stem Cells and Regeneration in Planarians
Fred Gage, The Salk Institute
Stem Cells in the Adult Nervous System
OCR for page 112
Sunday, October 20
Regeneration I: Organogenesis
Chair, Ken Chien
Margaret Fuller, Stanford University
Regulation of Stem Cell Turnover in Drosophila
Fiona Watt, Ca ncer Resea rch U K
Manipulation of the Stem Cell Compartment in Mammalian Epidermis
Markus Grompe, Oregon Health Sciences University
Liver Repopulation by Bone Marrow Derived Stem Cells
Juan Carlos Izpisua Belmonte, The Salk Institute
Regeneration and Notch Signaling
Owen Witte, University of California, Los Angeles
Prostate Development
Regeneration Il: Tissue Engineering
Chair, Shu Chien
George Daley, Whitehead Institute
Hematopoietic Engraftment from Embryonic Stem Cells Enables Therapeutic Cloning in
a Murine Model of Immunodeficiency
Ian Wilmut, Roslin Institute
Cloning
Jose Cibelli, Advanced Cell Technology
Parthenogenesis and Therapeutic Cloning
Darwin J. Prockop, Tulane University School of Medicine
Adult Stem Cells from Bone Marrow Stroma: Isolation, Characterization and Use in an
Ex Viva Assay for Repair of Epithelium
Peter Mombaerts, The Rockefeller University
Cloning and Embryonic Stem Cells
Representative terms from entire chapter:
cell lines
