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Wound Repair and Regeneration

Stem cells isolated from adult rat muscle differentiate across


all three dermal lineages
Sherri S. Schultz, PhD1n; Sherly Abraham, MD2; Paul A. Lucas, MD, PhD1,2

1. Department of Pathology, and


2. Department of Orthopaedic Surgery, New York Medical College, Valhalla, New York

Reprint requests: ABSTRACT


Paul Lucas, PhD, Department of
Orthopaedic Surgery, New York Medical Adult stem cells capable of differentiating into phenotypes from all three dermal
College, Macy Pavilion, Valhalla, NY 10595. layers were isolated from adult rat muscle. Stem cells were obtained by enzymatic
Email: Paul_Lucas@nymc.edu digestion, followed by primary culture in Eagle’s minimum essential medium
1 10% preselected horse serum. When the cells reached confluence, they were re-
n
Current address: Department of Cell leased by trypsin, filtered to remove differentiated myotubes, and then slow fro-
Biology, Albert Einstein College of zen in 7.5% dimethylsulfoxide to  80 1C. Thawed cells were the stem cells and
Medicine, Bronx, New York. were induced to differentiate with the nonspecific differentiating agent dexameth-
asone at concentrations of 10  10–10  6 M. After a 6-week treatment with dexa-
Manuscript received: December 24, 2004 methasone, the cells were assayed by immunohistochemistry for phenotypes of
Accepted in final form: October 21, 2005 the mesodermal, ectodermal, and endodermal lineages. Examples of mesodermal
phenotypes identified were as follows: bone, cartilage, and skeletal, smooth, and
DOI:10.1111/j.1743-6109.2006.00114.x cardiac muscle. Ectodermal phenotypes identified were as follows: neurons and
oligodendrocytes. Hepatocyte phenotypes identified represented the endodermal
lineage. All the phenotypes were observed only with treatment with dexametha-
sone. However, nestin was observed in the absence of dexamethasone and may be
a marker for uncommitted pluripotent stem cells. The results show that adult
muscle contains pluripotent stem cells capable of differentiating across all three
dermal lineages. Such cells could be used in the context of tissue engineering.

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.

Rat adult stem cells were isolated from 11–12-week-old


male F344 or Sprague–Dawley rats as described previous-
ly.5 The animals were euthanized in accordance with the AFP a-Fetoprotein
guidelines of the New York Medical College Institutional EMEM Earle’s minimum essential medium
Animal Care and Use Committee and the gluteal muscle PBS Phosphate buffered saline solution
was dissected.5 SSEA Stage-specific embryonic antigen

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Schultz et al. Stem cells from adult rat muscle

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

Dexamethasone concentration (M)

Antibodies 0 10  10 10  9 10  8 10  7 10  6

IA4 0 0 0 1–5 5–15 15–25


MF-20 0 0 1–5 1–5 1–5 0
Desmin 0 1–5 1–5 5–15 1–5 5–15
CIICI 0 0 1–5 0 1–5 1–5
WVIDI 0 1–5 0 1–5 0 0
MPIII 0 0 1–5 0 1–5 1–5
a-Sarcomeric actin 1–5
Troponin I 1–5 1–5
Stro-1 1–5 1–5
P4A4 5–15 1–5
RT-97 0 1–5 5–15 0 0 0
Rat 401 > 25 15–25 5–15 1–5 1–5 0
CNPase 0 5–15 1–5 1–5 0 0
OV-1 0 0 1–5 1–5 0 0
OV-6 0 0 0 1–5 1–5 0
AFP 0 15–25 15–25 15–25 15–25 15–25

Approximately 20,000 cells were used per well.

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Stem cells from adult rat muscle Schultz et al.

Figure 1. Muscle phenotypes


in pluripotent cell cultures iso-
lated from adult rat muscle and
treated with dexamethasone at
indicated concentrations for 4–6
weeks. Cultures were stained
with antibodies specific for the
muscle phenotypes. (A) 10–6 M
dexamethasone; anti-desmin.
(B) 10–8 M dexamethasone; an-
ti-desmin. (C) 10–9 M dexamet-
hasone; antimyosin heavy chain
(skeletal muscle). (D) 10–7 M
dexamethasone; anti-smooth
muscle a-actin (smooth mus-
cle). (E) 10–6 M dexamethasone;
cardiac sarcomeric myosin. (F)
10–6 M dexamethasone; tropo-
nin I (cardiac myocyte)—original
magnification  100. (All except
F: Original magnification  160)

dexamethasone showed a distinct differentiation com- Ectodermal cell lineage


pared with the control and the other dexamethasone con-
centrations (Figure 1A and B). Cells stained for the heavy Antibodies specific for neurofilaments stained cells in
chain of myosin were observed in cultures treated with 10  10 and 10  9 M dexamthasone (Figure 3A and B).
10  9 and 10  8 M dexamethasone (Figure 1C). Cells that Cells positive for CNPase, which stains for oligodendro-
stained with the antibody to smooth muscle were present cytes, were seen in cultures treated with higher doses,
at 10  6 and 10  7 M dexamethasone (Figure 1D), while 10  7 M, of the steroid (Figure 3C).
cells stained with sarcomeric myosin, indicative of cardiac
myocytes, were seen in 10  9 and 10  8 M dexamethasone Endodermal cell lineage
(Figure 1E). Cells positive for Troponin I, also indicative
of cardiac myocytes, were found in 10  7 and 10  6 M de- Three different antibodies for liver parenchymal cells,
xamethasone (Figure 1F). OV1, OV6, and AFP, bound to cell cultures treated with
The expression of cartilage and bone phenotypes dexamethasone. Hepatocytes were also detected by posi-
from muscle stem cells was consistent with previous re- tive staining for a-fetoprotein across all dexamethasone
sults.5 Nodules of cells that were positive for type II colla- concentrations except the controls (Figure 4A). The OV-6
gen were found in cultures treated with 10  6 or 10  9 M antibody positively stained cells in cultures treated with
(Figure 2A). Nodules of cells with a dense extra- 10  10 M dexamethasone (Figure 4B), while OV-1 posi-
cellular matrix positive for osteopontin and bone sialopro- tively stained cells in cultures treated with 10  9 M dexa-
tein were found in all dexamethasone concentrations methasone (Figure 4C).
tested (Figure 2B and C). Expression of CD34 positive
bone marrow stromal cells were seen at 10  10 and 10  9 M Nestin-positive cell staining
dexamethasone (Figure 2D). The anti-endothelial cell
marker stained selected cells at 10  6 M dexamethasone The glial progenitor cell antibody nestin stained most of
(Figure 2E). the cells in control cultures without dexamethasone

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Schultz et al. Stem cells from adult rat muscle

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

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Stem cells from adult rat muscle Schultz et al.

Figure 3. Ectodermal phenotypes in pluripotent cell cultures


isolated from adult rat muscle and treated with dexamethasone Figure 4. Endodermal phenotype in pluripotent cell cultures
at indicated concentrations for 4–6 weeks. Cells from the treat- isolated from adult rat muscle and treated with dexamethasone
ed cultures were stained with antibodies specific for neural at indicated concentrations for 4–6 weeks. Cells from the treat-
phenotypes. (A) 10–10 M dexamethasone; anti-neurofilament ed cultures were stained with antibodies for hepatocytes. (A)
(neuron). (B) 10–9 M dexamethasone; anti-neurofilament. (C) 10–10 M dexamethasone; anti-AFP. (B) 10–9 M dexamethasone;
10–7 M dexamethasone; anti-CNPase (oligodendrocyte). (Orig- OV-6. (C) 10–9 M dexamethasone; OV-1. (Original magnification
inal magnification  150)  100)

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

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Schultz et al. Stem cells from adult rat muscle

Figure 5. Nestin staining in


pluripotent cell culture isolat-
ed from adult rat muscle and
treated with dexamethasone
at indicated concentrations for
4–6 weeks. Cultures were
stained with anti-nestin. (A) 0
M dexamethasone. (B) 10–10
M dexamethasone. (C) 10–8 M
dexamethasone. (D) 10–6 M
dexamethasone. (Original mag-
nification  100)

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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REFERENCES the extracellular domain of endoglin using bacterially ex-


pressed recombinant fragments. Tissue Antigens 1997; 50:
1. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells 265–76.
migrate throughout forebrain and cerebellum, and they dif- 18. Solter D, Knowles BB. Monoclonal antibody defining a
ferentiate into astrocytes after injection into neonatal mouse stage-specific mouse embryonic antigen. Proc Natl Acad Sci
brains. Proc Natl Acad Sci USA 1999; 96: 10711–6. USA 1978; 75: 5565–9.
2. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan 19. Damjanov I, Fox N, Knowles BB, Solter D, Lange PH,
AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone Fraley EE. Immunohistochemical localization of murine
marrow as a potential source of hepatic oval cells. Science stage specific embryonic antigens in human testicular germ
1999; 284: 1168–70. cell tumors. Am J Pathol 1982; 108: 225–30.
3. Prockop DJ. Marrow stromal cells as stem cells for non-hem- 20. Kannagi R, Cochran NA, Ishigami F, Hakomori S, Andrews
atopoietic tissues. Science 1997; 276: 71–4. PW, Knowles BB, Solter D. Stage-specific embryonic anti-
4. Young HE, Ceballos EM, Smith JC, Mancini ML, Wright gens (SSEA-3 and -4) are epitopes of a unique globo-series
RP, Ragan BL, Bushell I, Lucas PA. Pluripotent mesencymal ganglioside isolated from human teratocarcinoma cells.
stem cells reside within avain connective tissue matrices. In EMBO J 1983; 2: 2355–61.
Vitro Cell Dev Biol Anim 1993; 29A: 723–36. 21. Dunsford H, Sell S. Production of monoclonal antibodies to
5. Lucas PA, Calcutt AF, Southerland SS, Wilson JA, Harvey preneoplastic liver cell populations induced by chemical car-
RL, Warejcka D, Young HE. A population of cells resident cinogens in rats, and to transplantable Morris hepatomas.
within embryonic and newborn rat skeletal muscle is capable Cancer Res 1989; 49: 4887–93.
of differentiating into multiple mesodermal phenotypes. 22. Dunsford HA, Karnasuta C, Hunt JM, Sell S. Different lin-
Wound Rep Reg 1995; 3: 449–60. eages of chemically induced hepatocellular carcinoma in rats
6. Debus E, Weber K, Osborn M. Monoclonal antibodies to defined by monoclonal antibodies. Cancer Res 1989; 49:
desmin, the muscle-specific intermediate filament protein. 4894–900.
EMBO J 1983; 2: 2305–12. 23. Grigoriadis AE, Heersche JN, Aubin JE. Differentiation of
7. Sprinkle TJ, Agee JF, Tippins RB, Chamberlain CR, Faguet muscle, fat, cartilage, and bone from progenitor cells present
GB, De Vries GH. Monoclonal antibody production to hu- in a bone-derived clonal cell population: effect of dexameth-
man and bovine 2 0 :3 0 -cyclic nucleotide 3 0 -phosphodiesterase asone. J Cell Biol 1998; 106: 2139–51.
(CNPase): high-specificity recognition in whole brain acetone 24. Saatcioglu F, Claret FX, Karin M. Negative transcriptional
powders and conservation of sequence between CNP1 and regulation by nuclear receptors. Semin Cancer Biol 1994; 5:
CNP2. Brain Res 1987; 426: 349–57. 347–59.
8. Bader D, Masaki T, Fischman DA. Immunochemical analy- 25. Burcin M, Kohne AC, Runge D, Steiner C, Renkawitz R.
sis of myosin heavy chain during avian myogenesis in vivo Factors influencing nuclear receptors in transcriptional re-
and in vitro. J Cell Biol 1982; 95: 763–70. pression. Semin Cancer Biol 5: 337–46.
9. Taylor CR. Immunoperoxidase techniques. Practical and 26. Adcock IM. Glucocorticoid-regulated transcription factors.
theoretical aspects. Arch Pathol Lab Med 1978; 102: 113–21. Pulm Pharmacol Ther 2001; 14: 211–9.
10. Holmdahl R, Rubin K, Klareskog L, Larsson E, Wigzell H. 27. Wolf G. The molecular mechanism of the stimulation of ad-
Characterization of the antibody response in mice with type ipocytes differentiation by a glucocorticoid. Nutr Rev 1999;
II collagen-induced arthritis, using monoclonal anti-type II 57: 324–6.
collagen antibodies. Arthritis Rheum 1986; 29: 400–10. 28. Dieudonne SC, Kerr JM, Xu T, Sommer B, DeRubeis AR,
11. Kasugai S, Nagata T, Sodek J. Temporal studies on the tissue Kuznetsov SA, Kim IS, Gehron Robey P, Young MF. Dif-
compartmentalization of bone sialoprotein (bsp), osteopon- ferential display of human marrow stromal cells reveals
tin (opn), and sparc protein during bone formation in vitro. J unique mRNA expression patterns in response to dexameth-
Cell Phys 1992; 152: 467–77. asone. J Cell Biochem 1999; 76: 231–43.
12. Gorski JP, Griffin D, Dudley G, Stanford C, Thomas R, 29. de Haij S, Adcock IM, Bakker AC, Gobin SJ, Daha MR, van
Huang C, Lai E, Karr B, Solursh M. Bone acidic glycopro- Kooten C. Steroid-responsiveness of renal epithelial cells:
tein-75 is a major synthetic product of osteoblastic cells and dissociation of trans-repression and trans-activation. J Biol
localized as 75- and/or 50-kDa forms in mineralized phases Chem 2003; 278: 5091–8.
of bone and growth plate and in serum. J Biol Chem 1990; 30. Hockfield S, Kalb KG, Zaremba S, Fryer H. Expression of
265: 14956–63. neural proteoglycans correlates with the acquisition of ma-
13. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells ture neuronal properties in the mammalian brain. Cold
express a new class of intermediate filament protein. Cell Spring Harb Symp Quant Biol 1990; 55: 505–14.
1990; 60: 585–95. 31. Warejcka DJ, Harvey R, Taylor BJ, Young HE, Lucas PA. A
14. Friedman B, Zaremba S, Hockfield S. Monoclonal antibody population of cells isolated from rat heart capable of differ-
Rat 401 recognizes Schwann cells in mature developing entiating into several mesodermal phenotypes. J Surg Res
peripheral nerve. J Comp Neurol 1990; 295: 43–51. 1996; 62: 233–42.
15. Wood JN, Anderton BH. Monoclonal antibodies to mam- 32. Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat
malian neurofilaments. Biosc Rep 1981; 1: 263–8. and human bone marrow stromal cells differentiate into neu-
16. Friedman B, Hockfield S, Black JA. In situ demonstration of rons. J Neurosci Res 2000; 61: 364–70.
mature oligonucleotides and their processes on immunocyto- 33. Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi
chemistry study with a new monoclonal antibody, RIP. Glia AL. Turning brain into blood: a hematopoietic fate adopted
1989; 2: 380–90. by adult neural stem cells in vivo. Science 1999; 283: 534–7.
17. Pichuantes S, Vera S, Bourdeau A, Pece N, Kumar S, Way- 34. Romero-Ramos M, Vourc’h P, Young HE, Lucas PA, Wu Y,
ner EA, Letarte M. Mapping epitopes to distinct regions of Chivatakarn O, Zaman R, Dunkelman N, el-Kalay MA,

230 Wound Rep Reg (2006) 14 224–231 


c 2006 by the Wound Healing Society
Schultz et al. Stem cells from adult rat muscle

Chesselet MF. Neuronal differentiation of stem cells isolated 46. Strutz F, Muller GA. Transdifferentiation comes of age.
from adult muscle. J Neurosci Res 2002; 69: 894–907. Nephrol Dial Transplant 2000; 15: 1729–31.
35. Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Ver- 47. Condorelli G, Borello U, De Angelis L, Latronico M, Sira-
faillie CM. Multipotent progenitor cells can be isolated from bella D, Coletta M, Galli R, Balconi G, Follenzi A, Frati G,
postnatal murine bone marrow, muscle, and brain. Exp Hem- Cusella De Angelis MG, Gioglio L, Amuchastegui S, Adorini
atol 2002; 30: 896–904. L, Naldini L, Vescovi A, Dejana E, Cossu G. Car-
36. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene diomyocytes induce endothelial cells to transdifferentiate in-
CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, to cardiac muscle: implications for myocardium regenera-
Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaes- tion. Proc Nat Acad Sci USA 2001; 98: 10733–8.
pada DA, Verfaillie CM. Pluripotency of mesenchymal stem 48. Suzuki A, Zheng Yw YW, Kaneko S, Onodera M, Fukao K,
cells derived from adult marrow. Nature 2002; 418: 41–9. Nakauchi H, Taniguchi H. Clonal identification and charac-
37. Thomson JA, Kalishman J, Golos TG, Durning M, Harris terization of self-renewing pluripotent stem cells in the devel-
CP, Hearn JP. Pluripotent cell lines derived from common oping liver. J Cell Biol 2002; 156: 173–84.
marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996; 49. Jackson KA, Mi T, Goddell MA. Hematopoietic potential of
55: 254–9. stem cells isolated from murine skeletal muscle. Proc Natl
38. Thomson JA, Kalishman J, Golos TG, Durning M, Harris Acad Sci USA 1999; 96: 14482–6.
CP, Becker RA, Hearn JP. Isolation of a primate embryonic 50. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita
stem cell line. Proc Natl Acad Sci USA 1995; 92: 7844–8. Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF,
39. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Hammond WP. Evidence for circulating bone marrow-
Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell derived endothelial cells. Blood 1998; 92: 362–7.
lines derived from human blastocysts. Science 1998; 282: 51. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ,
1145–7. Majesky MW, Entman ML, Michael LH, Hirschi KK, Goo-
40. Martin GR. Isolation of a pluripotent cell line from early dell MA. Regeneration of ischemic cardiac muscle and vas-
mouse embryos cultured in medium conditioned by terato- cular endothelium by adult stem cells. J Clin Invest 2001; 107:
carcinoma stem cells. Proc Natl Acad Sci USA 1981; 78: 1395–402.
7634–8. 52. Brazelton TR, Rossi FMV, Keshet GI, Blau HM. From mar-
41. Park TS, Han JY. Derivation and characterization of pluri- row to brain: expression of neuronal phenotypes in adult
potent embryonic germ cells in chicken. Mol Reprod Dev mice. Science 2000; 290: 1775–9.
2000; 56: 475–82. 53. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM,
42. Vassilieva S, Guan K, Pich U. Establishment of SSEA-1- and Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri
Oct-4-expressing rat embryonic stem-like cell lines and effects A, Anversa P. Bone marrow cells regenerate infracted
of cytokines of the IL-6 family on clonal growth. Exp Cell myocardium. Nature 2001; 410: 701–5.
Res 2000; 258: 361–73. 54. Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F,
43. Orkin SH, Zon LI. Hematopoiesis and stem cells: plasticity Sadikot A, Kaplan DR, Miller FD. Isolation of multipotent
versus development heterogeneity. Nat Immunol 2002; 3: adult stem cells from the dermis of mammalian skin. Nat Cell
323–8. Biol 2001; 3: 778–84.
44. Tosh D, Slack JMW. How cells change their phenotype. Mol 55. Fuchs E, Segre JA. Stem cells: a new lease on life. Cell 2000;
Cell Biol 2002; 3: 187–94. 100: 143–55.
45. Bouwens L. Transdifferentiation versus stem cell hypothesis 56. Bonassar LJ, Vacanti CA. Tissue engineering: the first
for the regeneration of islet beta-cells in the pancreas. Microsc decade and beyond. J Cell Biochem 1998; 30 (Suppl):
Res Tech 1998; 15: 332–6. 297–303.

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