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Stem cells are self-renewing cells and generate progeny Briefly, the muscles were minced with curved scissors
that are capable of differentiating into a cell lineage.1 Stem and the minced muscle was transferred to a 100 mL sterile
cells derived from early embryos can differentiate into all bottle with a stir bar. The muscle was then enzymatically
somatic cell types, and some populations of adult stem digested with collagenase/dispase at a ratio of tissue: Ea-
cells existing within various parts of the body may function gle’s minimum essential medium (EMEM; Life Technolo-
similarly. For example, stem cells isolated from the bone gies, Long Island, NY) : EMEM 1 3% bovine serum
marrow give rise to hepatic cells and mature astrocytes and albumin : collagenase/dispase of 1 : 13 : 2 : 4 with a final
neurons.1–3 concentration of collagenase/dispase of 250 U/mL colla-
We have previously demonstrated that muscle contains genase and 33 U/mL dispase. All solutions had antimy-
a population of mesenchymal stem cells that are capable of cotic–antibiotic solution added at 1% concentration. The
differentiating into many of the phenotypes in the meso- tissue was digested at 37 1C with stirring for 1–2 hours. The
dermal lineage.4,5 In addition to the identified phenotypes, cell suspension was filtered through a 20 mM Nitex filter
there were unidentified morphologies in the cultures treat- (Argent Labs, Redmond, WA) and then centrifuged at 300
ed with dexamethasone. New antibodies against specific g for 30 minutes. The cells were resuspended in EMEM
proteins of ectodermal and endodermal phenotypes have with 10% preselected horse serum (Gem Cell, Woodland,
become available and, therefore, in this study we tested CA, lot E73104R) and plated at a density of 1 105 cells
whether dexamethasone would induce phenotypes of all per 100 mm gelatin-coated culture dish.
three dermal lineages. This would indicate whether the dif- The cultures were maintained in EMEM with 10% pre-
ferentiation capability of this population of stem cells in- selected horse serum until the cell layer was confluent. The
cludes all three dermal lineages. cultures were then trypsinized and filtered through a 20
mM filter to remove the myotubes. The cells were cryopre-
served at 106 cells in EMEM with preselected 10% horse
MATERIALS AND METHODS serum and 7.5% dimethysulfoxide (Sigma-Aldrich, St.
Louis, MO) and frozen to 80 1C in a freezing chamber Primary antibodies used for these studies included the
(Fisher Scientific, Norcross, GA). After at least a 24-hour following: muscle-type intermediate filaments with mono-
period, cells were thawed and plated at a density of 10,000 clonal anti-desmin clone De-U-10 (Sigma-Aldrich, cat.
cells per well in 24-well gelatin-coated plates (Corning No. D1033) 1 : 2006; anti-CNPase (Sigma-Aldrich, cat.
Glass Works, Corning, NY). The next day, the cells were No. C5922) 1 : 2007; anti-cardiac troponin I (Advanced
treated with concentrations of dexamethasone (Sigma- ImmunoChemical Inc., Long Beach, CA); heavy chain of
Aldrich) in EMEM with 10% horse serum—0 (control), myosin, MF-20,8 smooth muscle a-actin (IA4),9 type II
10 10, 10 9, 10 8, 10 7, and 10 6 M. The cells were collagen (CIICI),10 bone sialoproteins I and II, WVIDI
fixed after 6 weeks.5 9C511; osteopontin, MPIIIB10112; spinal cord, Rat-
40113,14; neurofilaments, RT-9715; early oligodendrocytes,
RIP16; endothelial cells, P4A4; and 17 stage-specific em-
Assays for phenotypes bryonic antigen (SSEA)-1,3,4, MC-480, MC-631, MC-
The presence of phenotypes was determined by antibody 813-70,18–20 all from the Developmental Studies Hybrid-
labeling of the cell cultures in 24-well plates. Cells were oma Bank (Iowa City, IA) and used at a dilution of 1 : 200.
washed three times in phosphate buffered saline solution a-Fetoprotein (AFP) was from DAKO Corp. (Car-
(PBS), followed by fixing with 20 1C methanol. After a pinteria, CA). OV-6, a cytokeratin antibody, and OV-1,
further three PBS rinses, the cells were treated with 0.4 mL an uncharacterized membrane antigen, were both liver an-
of 0.3% H2O2 in a 0.1% Triton X-100 (Sigma, St. Louis, tibodies created by Dunsford and Sell.21,22 The monoclon-
MO) in PBS for 10 minutes. This solution was removed, al antibodies developed by Michael Solursh and Ahnders
the cells were rinsed three times with PBS, and the cells Frazen (WV1D1 9C5, CIICI, MF-20, MPIIIB101), Susan
were exposed to 0.4 mL of 1% goat serum in a 0.5% Hockfield (Rat-401), John Wood (RT-97), Elizabeth A.
Tween-20 (Sigma) prepared in PBS for 30 minutes at Wayner (P4A4), and Davor Salter SSEA-1,3,4 (MC-480,
37 1C, which served to block nonspecific binding sites. Af- MC-631, MC-813-70) were obtained from the Develop-
ter a further three PBS rinses, 0.3 mL of the primary anti- mental Studies Hybridoma Bank. Stro-1 was developed by
body in PBS with 0.1% Triton X-100 was added for 60 Beverly Torok-Storb at the Fred Hutchinson Research
minutes at 37 1C. The antibody was removed by washing Center. OV-1, AFP, and OV-6 were generous gifts from
the cells three times with PBS containing Triton X-100. Dr. Stewart Sell.
The cells were again treated with 0.4 mL of 1% goat serum
in PBS with 0.5% Tween for 20 minutes, and this was fol- RESULTS
lowed by 0.3 mL of IgG-HRP secondary antibody (KPL,
Gaithersburg, MD) in PBS with 0.1% Triton X-100 dilut- Muscle phenotypes were seen across all of the dexameth-
ed 1 : 2,000 incubation for 30 minutes at 37 1C. The cells asone concentrations except the controls. The anti-desmin
were rinsed five times with PBS, followed by 0.3 mL of antibody showed muscle in 10 6, 10 7 10 8, 10 9, and
True Blue (KPL), and light protected for up to 20 minutes. 10 10 M, with the highest number of cells staining in 10 6
Finally, the cells were rinsed five times with distilled H2O. M dexamethasone (Table 1). The stained cells in 10 6 M
Table 1. Number of cells/well stained with antibodies and the respective dexamethasone concentrations
Antibodies 0 10 10 10 9 10 8 10 7 10 6
Figure 2. Mesodermal
phenotypes in pluripotent
cell cultures isolated from
adult rat muscle and treated
with dexamethasone at indi-
cated concentrations for 4–6
weeks. Cells from the treated
cultures were stained with an-
tibodies of the mesodermal
phenotype. (A) 10–10 M dexa-
methasone; anti-type II colla-
gen (cartilage). (B) 10– M
dexamethasone; anti-type II
collagen. (C) 10–6 M dexameth-
asone; antibone sialoprotein
(bone). (D) 10–9 M dexameth-
asone; anti-CD34 (stromal
cell). (E) 10–6 M dexameth-
asone; anti-endothelial cell.
(Original magnification 160)
(Figure 5A). The number of cells staining for nestin de- DISCUSSION
creased as dexamethasone concentration increased (Figure
5B–D). No other neuronal markers stained cells in the ab- Glucocorticoids have long been recognized as inducers of
sence of dexamethasone; therefore, it is possible that nestin differentiation even though the mechanism action is still
is a marker for these stem cells. The cultures were also not fully elucidated; it may involve direct initiation of
tested for the presence of SSEA 1, 3, and 4, known markers transcription of genes in some cases and derepression of
for embryonic stem cells. All concentrations of dexa- genes in other cases.24–29 Dexamethasone differentiated
methasone including controls were negative for these anti- the stem cells into all three of the dermal lineages, alleviat-
gens (data not shown). ing the need for individual culture conditions for differen-
The overall staining pattern (Table 1) shows that tiation into each lineage. Dexamethasone was used solely
phenotypes appeared in two or more dexamethasone con- to assay for multipotency.
centrations but rarely in all the concentrations. The major Dexamethasone treatment induced the differentiation
exceptions were bone nodules and AFP. The pattern of of several mesodermal phenotypes: striated muscle,
cells positive for osteoblast markers is consistent with pre- smooth muscle (smooth muscle a-actin), cartilage (type II
vious reports.5,23 In general, ectodermal and endodermal collagen), bone (bone sialoproteins I and II and osteonec-
phenotypes appeared in cultures treated with less than tin), and adipocytes (Sudan Black B, Sigma, data not
10 7 M dexamethasone. Some mesodermal phenotypes, shown) (Figures 1 and 2). The specific phenotypes ap-
such as smooth muscle and fat, appear in cultures treated peared at a range of concentrations of dexamethasone that
with greater than 10 9 M dexamethasone while others ap- correlated with previous studies.5 This suggests that the
peared in cultures treated at the two lower dexamethasone cells isolated in this study were identical to the cells iso-
concentrations. lated in previous studies. However, two additional
mesodermal phenotypes were identified in this study, car- None of the antibodies mentioned above stained adult
diac muscle (troponin-I), and bone marrow stromal fibro- stem cells that were not treated with dexamethasone, indi-
blasts (stro-1). cating that the cultures did not contain precursor cells for
This population of cells also differentiated into pheno- these phenotypes. The one exception to this overall finding
types of the ectodermal and endodermal lineages as deter- was an antibody to nestin (Rat 401). Nestin has been re-
mined by specific antibody staining. Examples from the ported to be a glial progenitor marker in neural stem cells
ectodermal lineage include oligodendrocytes (CNPase) isolated from the brain,13,30 but it is unlikely that neural
and neurons (RT-97). The neuronal phenotypes were stem cells would be present in skeletal muscle. We hypoth-
mainly induced by lower dexamethasone concentrations. esize that nestin is a marker not only for committed neural
We only tested for induction of one endodermal tissue: stem cells but also for the more primitive stem cells isolat-
liver. A varying range of dexamethasone concentrations ed in this study. Further studies on adult stem cells isolated
had cells positive for the hepatocyte antibodies. OV-1- and from different tissues and different species will test this
OV-6-positive cells were observed in 10 9–10 7 M dexa- hypothesis.
methasone. Cells positive for AFP were observed in all Other studies on adult stem cells have used specific cul-
cultures treated with dexamethasone. ture conditions to differentiate the cells to each specific
phenotype.31–36 This raises the possibility that the culture contained uncommitted stem cells that responded to sig-
conditions may selectively promote the survival and pro- nals in the new tissue to differentiate into phenotypes of
liferation of individual progenitor cells in the cultures and the new tissue. Three studies report the in vitro differenti-
do not detect the existence of a more primitive stem cell. In ation of stem cells into phenotypes from dermal lineages
our studies, the general culture conditions were the same other than the source of the stem cells.33,36,54 This suggests
for each phenotype induced: a monolayer culture in a more primitive stem cell that is uncommitted to a partic-
EMEM 1 10% preselected serum, 37 1C, and 5% CO2. In- ular lineage. Our results are in accord with these results.36
duction of each phenotype by dexamethasone occurred in Multinucleated myotubes appear in the primary cultures.
at least two concentrations, and at least two phenotypes This finding represents the differentiation of committed
appeared at each concentration of dexamethasone (Table myogenic progenitor cells—satellite cells. However, filtra-
1). These observations make it unlikely that the results are tion of these primary cultures removes all the myotubes
due to a population of mixed progenitor cells. and freeze–thawing results in a culture composed solely of
Although we detected phenotypes from all three of the mononucleated cells. These cells do not form multinucle-
dermal lineages, the dozen phenotypes detected in this ated myotubes unless induced with the appropriate con-
study are obviously not all of the more than 200 pheno- centration of dexamethasone. Similarly, smooth muscle
types known to exist in the adult mammal. However, so and endothelial cells are also absent from cell preparations
far, we have detected every phenotype tested. Also, the after the freeze–thaw; yet, both progenitor cell types are
dozen phenotypes induced here have also been reported to present in the primary culture. If the cultures did contain
differentiate in cultures of embryonic stem cells.37–40 progenitor cells that were transdifferentiating, these phe-
Therefore, it is possible that the differentiation potential notypes normally present in skeletal muscle should be seen
of the adult stem cells may be equivalent to that of embry- in the absence of the dexamethasone treatment. Their ab-
onic stem cells. It is unlikely that the adult stem cells are sence supports the hypothesis that this is a resident popu-
embryonic stem cells persisting into the adult organism. lation of primitive uncommitted adult stem cells that
The adult stem cells were negative for SSEA 1, 3, and 4, retain the ability to commit to phenotypes of all three der-
while all reported embryonic stem cells are positive for at mal lineages when exposed to differentiating agents.
least one of these antigens.41,42 The differentiation poten- The existence of a population of very primitive stem
tial of these adult stem cells will continue to be explored as cells that can be isolated from adult muscle and expanded
markers for specific phenotypes become available. in vitro has promising implications for regenerative med-
The ability of cells to cross lineages is known as plastic- icine. The application of these endogenous adult stem cells
ity. One theory of cellular plasticity, or transdifferentia- could treat degenerative diseases, diabetes, and organ fail-
tion, states that cells from one developmental lineage can ure by regenerating damaged tissues.55,56
reprogram genetically to differentiate into phenotypes
from a different lineage.43–47 Most of the studies support-
ing this theory are in vivo studies using populations of ACKNOWLEDGMENTS
cells.48–53 Although the data are interpreted in that tissue-
specific stem cells transdifferentiate into cells of another This work was funded by grants from New York Medical
lineage, it is equally possible that the cell preparations College.
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