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Comment on "Failure of Bone Marrow Cells to Transdifferentiate into Neural Cells in Vivo"
Castro et al. (1) reported
that bone marrow cells (BMCs) fail to generate neural cells in vivo.
The bone marrow theyinjected came from mice in which LacZ expression
was driven bya widely expressed trapped promoter. In principle, such
cellsand their progeny should readily be detected by staining for
-galactosidaseactivity. To validate their method, Castro et
al. presented twobrain sections, one completely white
(transplanted mouse) andone completely blue (donor). The problem with
these results, however,is that blue LacZ- positive
microglia--which, like other monocyte/macrophagecells, originate from
hematopoietic stem cells [see (2)]--wereabsent from the
brains of the transplanted animals.
In contrast, several investigators have shown that BMCs
expressing enhanced green fluorescent protein (EGFP) under the controlof a similarly widespread promoter give rise to EGFP-positivemicroglia
in the brains of transplanted animals (3-5).Nakano
et al. (6) and Eglitis and Mezey
(7)obtained similar results using a retroviral
insert or the Y chromosome,respectively, as markers of cell lineage.
One might argue that Castro et al. did not
wait long enough to see tagged microglia, but approximately 6% of the
microglialcell population is replaced per month (3, 8).Thus, they should have detected LacZ in 6 to 24% of the microgliapresent in the brains of their animals, equivalent to 500 to 1000positive cells per section at the shortest time (1 month) studied.Their failure to detect LacZ-positive cells indicates that eithertheir
staining method failed or that the LacZ transgene was notexpressed in
their samples.
We noticed a similar result in irradiated female mice
transplanted with EGFP-expressing male marrow cells. There were manymore Y chromosome-positive cells than green cells in the brain,even
when we used a very sensitive immunohistochemical techniqueto
visualize EGFP (Fig. 1). In peripheral tissues
(thymus,spleen, or bone marrow) we detected many bright green cells,
mostof which were also Y chromosome-positive (when the nucleus wasin
the section). In view of these results, we question the conclusionsin
(1), though we agree that the demonstration of bonemarrow-derived cells in the brain surely "depend[s] on the
experimentalsystem in which the hypothesis is tested." The system
used byCastro et al. (1) may not have been an
ideal one.
Fig. 1.
Discrepancy between EGFP expression and the
presence of Y chromosome in the CNS after gender mismatched EGFP bone
marrow transplantation. Coronal brain sections of a female mouse that
received a male EGFP bone marrow transplant one month earlier are
shown. (A) EGFP-derived fluorescence (FITC filter).
Except for a few cells in the choroid plexus (asterisk), there are
no green cells visible. (B) Photograph of the same
area using a rhodamine (red) fluorescence filter. Labeled Y chromosomes
appear as red dots (arrows). Note the numerous red dots in the brain
parenchyma (cortex and habenula) and the lack of green cells in the
same regions. Hb, habenula; Hc, hippocampus.
[View Larger Version of this Image (16K GIF file)]
The use of protein products of transgenes as markers to
follow graft fate is plagued with problems, but these may not havebeen
emphasized enough. It is virtually impossible to achieveubiquitous
transgene expression. Transgenes, including those drivenby the Rosa26
promoter, suffer from instability in several tissues(9).
Detection of low levels of LacZ is also difficult,and is sensitive to
fixation and staining conditions (9).Furthermore, reporter
expression might place cells at a growthdisadvantage compared with
cells without a reporter construct.Combined with instability of the
transgene, selection againstlabeled cells could make the vast
majority, if not all, of thegrafted cells "invisible."
Undoubtedly, reporters make graftingexperiments less tedious to
perform, but because of the presentuncertainties concerning transgenic
expression tagging, DNA markerssuch as Y chromosomal probes are far
more reliable.
We recently showed that bone marrow- derived cells
enter the brain and differentiate into neural cells in humans
(10)and that they give rise to cheek epithelial cells
without anyevidence of fusion (11). A similar report
confirmedthat mouse BMCs can indeed become neural cells and showed
thatthis happens without fusion (5). Thus, we feel that
adultstem cells may be induced to reconstitute other tissues than
thosefrom which they were harvested.
Éva Mezey
National Institute of
Neurological Disorders and Stroke Building 36, Room 3D-10 9000 Rockville Pike Bethesda, MD 20892, USA E-mail:
mezeye{at}ninds.nih.gov Andras Nagy
Mount Sinai Hospital Samuel
Lunenfeld Research Institute 600 University Avenue Toronto, ON
M5G 1X5, Canada Ildiko Szalayova Sharon Key
National
Institute of Neurological Disorders and Stroke Andras Bratincsak
National Institute of Mental
Health Bethesda, MD 20892, USA Judit Baffi
National Eye Institute Bethesda,
MD 20892, USA Tal Shahar
National Institute of Neurological Disorders and Stroke
10 October 2002; accepted 19 December
2002
10.1126/science.1079318 Include this information when citing this paper.
The editors suggest the following Related Resources on Science sites:
In Science Magazine
TECHNICAL COMMENTS
Raymond F. Castro, Kathyjo A. Jackson, Margaret A. Goodell, Claudia S. Robertson, Hao Liu, and H. David Shine (21 February 2003) Science299 (5610), 1184c.
[DOI: 10.1126/science.1080631] |Full Text »|PDF »
BREVIA
Raymond F. Castro, Kathyjo A. Jackson, Margaret A. Goodell, Claudia S. Robertson, Hao Liu, and H. David Shine (23 August 2002) Science297 (5585), 1299.
[DOI: 10.1126/science.297.5585.1299] |Full Text »|PDF »|Supporting Online Material »
-galactosidase activity. To validate their method, Castro et
al. presented two brain sections, one completely white
(transplanted mouse) and one completely blue (donor). The problem with
these results, however, is that blue LacZ- positive
microglia--which, like other monocyte/macrophage cells, originate from
hematopoietic stem cells [see (
