Wagers et al. (1) investigated the
possibility of marrow cell plasticity at the single cell level, as we
did (2). Although they found "little evidence for
developmental plasticity"-- i.e., marrow cells giving rise to
mesoderm (blood), endoderm (liver), and ectoderm (brain)--their
experiment substantially differed from ours and is therefore not a
clear refutation of our previous work.
Wagers et al. used 6- to 12-week-old donor and 10- to
14-week-old recipient mice. Our donor and recipient mice were
age-matched at 4 to 6 weeks old. Although age may not affect the rate
of hematopoietic engraftment, it may affect plasticity potential.
Hematopoietic stem cells (HSCs) have different phenotypes in younger (3 to 5 weeks) than in older (5 weeks) mice. For example, murine HSCs express the CD34 antigen in mice younger than 5 weeks, but not older
(3).
We analyzed engraftment 11 months following stem cell transplantation,
while Wagers et al. analyzed mice from 4 to 9 months after transplantation. It is unclear from (1) how many mice
were analyzed at different time points, how much donor- derived marrow engraftment each mouse had, and whether there was an association between time of sacrifice and presence of donor-derived hepatocytes or
Perkinje cells. For example, Wagers et al. reported that
their mice had 0.03 to 71.6% peripheral blood engraftment, but
engraftment levels in the four mice analyzed were not specified, nor
was the level of blood engraftment stated for the time that the solid tissues were examined.
More important, the differently purified stem cells likely
represent different rare subpopulations with functionally different levels of plasticity. We isolated and purified our stem cells through
functional isolation involving lineage depletion, elutriation, bone
marrow homing, and separation of small, G0/G1
cells. We have shown (4) that elutriated,
phenotypically negatively selected cells are enriched for stem cell
engraftment following a homing step to the marrow in vivo. In contrast,
Wagers et al. used purely phenotypic fluorescence-activated
cell sorting (FACS) to select for
c-kit+Thy1.1loLin-Sca-1+
(KTLS) cells.
The most important difference in methods may be our inclusion of AA4.1
antibody in our Lin cocktail, a step that removed hematopoietic progenitors and possibly allowed selection for an "earlier" stem cell with more plasticity potential. The parabiotic experiments performed by Wagers et al. would of course include the cell
population we tested, but they did not assess engraftment following
injury (including whole body or targeted irradiation) in that
model. Thus, the absence of evidence of plasticity in the parabiotic model may simply indicate the need for injury to elicit a plasticity response.
The methods used for engraftment detection may also differ in
sensitivity. We used the Y chromosome as a marker of marrow cell
origin. Any cell derived from the engrafted population, by any
mechanism (fusion or differentiation), would contain a Y chromosome. Wagers et al. used the green fluorescent protein (GFP)
transgene expressed from the chicken 
-actin promoter with a
cytomegalovirus (CMV) enhancer. Although this promoter was expressed in
all cell types tested, there is no evidence that all marrow cells would express GFP when and if they differentiated into nonhematopoietic cells. In addition, it is uncertain whether the level of any transgene in a newly "transdifferentiated" cell would always be detectable above background autofluorescence in different tissues. For
example, following transplantation of male Rosa marrow cells into
lethally irradiated female wild type mice, the spleen showed >90%
engraftment by Y chromosome analysis, but cells expressing
-galactosidase expression represented <50% of the splenic cells
(5). This is important, given that the Rosa mouse is
commonly said to show "ubiquitous" transgene expression, as
Wagers et al. claimed for their relatively
uncharacterized GFP+ transgenic mouse. It is possible that
transplantation and expansion of the donor-derived cell population
could affect transgene expression. Therefore, transgene expression,
even if driven by a presumed "ubiquitously" active promoter, is a
less rigorous and nonoptimal means for identifying donor-derived cells.
In summary, the Wagers et al. study differs significantly
from our prior work and clearly shows that further experimentation is
necessary to determine the factors that inhibit or promote adult cell
plasticity responses.
Neil D. Theise
Division of Digestive Diseases
Departments of Medicine and
Pathology
Beth Israel Medical Center
1st Avenue at 16th Street
New York, NY 10003, USA
Diane S. Krause
Yale University School of Medicine
Department of Laboratory
Medicine
Post Office Box 208035
New Haven, CT 06520-8035, USA
Saul Sharkis
Sidney Kimmel Comprehensive
Cancer Center
Johns Hopkins
University
BBCRB Room 244 1650 Orleans Street
Baltimore, MD
21231, USA
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11 September 2002; accepted 8 January
2003
10.1126/science.1078412
Include this information when citing this paper.
