Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 101
Colloquium
One strategy for cell and gene therapy: Harnessing
the power of adult stem cells to repair tissues
Darwin J. Prockop*, Carl A. Gregory, and Jeffe~y L. Spees
Center for Gene Therapy, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL-99, New Orleans, LA 70112
t,
Most recent evidence suggests that the process of tissue repair is
driven by stem-like cells that reside in multiple tissues but are
replenished by precursor cells from bone marrow. Among the
candidates for the reparative cells are the adult stem cells from
bone marrow referred to as either mesenchymal stem cells or
marrow stromal cells (MSCs). We recently found that after MSCs
were replated at very low densities to generate single-cell-derived
colonies, they did not exit a prolonged lag period until they
synthesized and secreted considerable quantities of Dickkopf-1, an
inhibitor of the canonical Wnt signaling pathway. We also found
that when the cells were cocultured with heat-shocked pulmonary
epithelial cells, they differentiated into epithelial cells. Most of the
MSCs differentiated without evidence of cell fusion but up to
one-quarter underwent cell fusion with the epithelial cells. A few
also underwent nuclear fusion. The results are consistent with the
interesting possibility that MSCs and similar cells repair tissue
injury by three different mechanisms: creation of a milieu that
enhances regeneration of endogenous cells, transdifferentiation,
and perhaps cell fusion.
One of the most intriguing questions in both biology and
medicine is: How do complex organisms repair injured
tissues? The repair of tissues in mammals is not as efficient as the
dramatic regeneration of whole body parts that is observed in
simple organisms such as planaria, hydra, or even vertebrates
such as newts (see ref. 14. At the same time, it is apparent that
tissue repair is an ongoing process throughout the lives of the
most complex organisms.
How Are Tissues Repaired? The Cohnheim Hypothesis
One hypothesis about tissue repair in mammals was advanced in
the middle of the 19th century by Cohnheim (2), who suggested
that all of the cells come from the bloodstream and therefore, in
light of subsequent observations, from the bone marrow.
Cohnheim's hypothesis has been the subject of much debate and
experimentation. Some early experiments in animal models
appeared to rule out the possibility that any of the noninflam-
matory cells involved in healing tissues and organ originated
from the bloodstream and bone marrow (see ref. 34. Therefore,
most studies on wound repair focused on cells resident in the
tissues such as pericytes that are seen to proliferate during the
repair in most tissues (see refs. 4 and 54. The interest in resident,
reparative cells has been heightened by the recent observations
that small, stem-like cells with considerable plasticity are found
in a variety of tissues including muscle (6, 7), fat (8), liver (9, 10),
synovial membranes (11), and brain (12~. At the same time,
recent observations have largely validated Cohnheim's hypoth-
esis and indicated that the stem cells found in most tissues are
replenished by stem cells for nonhematopoietic tissues found in
the marrow.
Reparative Cells from Bone Marrow: Data from
Experimental Animals
A large series of reports have indicated that marrow-derived cells
were found to engraft as differentiated cells into multiple tissues
www.pnas.org/cgi/cloi/10. ~ 073/pnas. ~ 834138100
after infusion into experimental animals of either whole bone
marrow or one or more subpopulations from marrow. The
number of such publications is now too large to summarize in the
present context. (For a partial list of the publications, see refs.
4 and 13-27.) Also, several reports have described isolation of
precursor cells for nonhematopoietic tissues from peripheral
blood (28-304. However, several recent publications have chal-
lenged some of the evidence that bone marrow contains cells that
can repair nonhematopoietic tissues.
Wagers et al. (31) transplanted single hematopoietic cells that
expressed GFP into lethally irradiated mice and examined
parabiotic mice in which the cells of one partner expressed GFP.
They concluded that there was "little evidence for developmen-
tal plasticity of adult hematopoietic stem cells." Their results
therefore seemed to challenge several publications reporting
that some hematopoietic stem cells can also differentiate in vivo
into nonhematopoietic cells (see refs. 23-254. As indicated by the
subsequent exchange of comments (32, 33), the apparent dis-
crepancies are probably explained by several subtle differences
in the experimental conditions. In particular, there were differ-
ences in the subpopulations of the cells used for the experiments
and in the sensitivity and the stability of the genes used to mark
the donor cells. A similar challenge came from the report by
Castro et al. (34), who followed Lc~cZ gene label in hematopoietic
stem cells, referred to as SP cells, after i.v. infusion into mice that
were lethally irradiated. They examined brains from the recipient
mice and interpreted their results as a "failure of bone marrow
cells to transdifferentiate into neural cells in vivo." Therefore the
results were apparently inconsistent with several previous re-
ports that identified donor-derived neural cells in mice after i.v.
infusion of bone marrow cells (15, 16, 21~. As again reflected in
an exchange of comments (35, 36), the apparent discrepancies
can probably be explained by a number of critical experimental
variables.
A further challenge to some of the conclusions about repar-
ative cells in bone marrow came from reports indicating that at
least some of the plasticity of adult stem cells can be attributed
to cell fusion and not direct differentiation (37-39~. Cell fusion
was observed when adult stem cells from the CNS (37) or
unfractionated mononucleated cells from marrow (38) were
added to cultured embryonic stem cells, and the added cells were
selected for by the presence of a selectable marker gene. The
resulting tetraploid cells exhibited full pluripotentiality, includ-
ing multilineage contribution to chimeras (374. In unrelated
experiments, bone marrow-derived hepatocytes were serially
This paper results from the Arthur M. Sackier Colioquium of the Nationai Acaclemy of
Sciences, "Regenerative Mecdicine," heicl October 18-22, 2002, at the Arnoicl ancl Mabei
Beckman Center of the National Academies of Science anc] Engineering in irvine, CA.
Abbreviations: MSC, mesenchymai stem ceil or marrow stromai ceil; RS ceils, rapiclly
self-renewing ceils; Dkk-1, Dickkopf-~; SAEC, smaii airway epitheiiai ceil.
*To whom corresponcience shouicl be aciciressecI. E-ma ii : ciprocko@tuia ne.eclu.
@) 2003 by The Nationai Acacdemy of Sciences of the USA
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11917-11923
OCR for page 102
transplanted into transgenic mice that are a model for tyrosine-
mia (40~. The results indicated that the marrow-derived hepa-
tocytes that rescued the mice arose by cell fusion and not by
differentiation.
At the moment it is difficult to resolve all of the apparent
discrepancies in observations on the presence of reparative cells
in bone marrow, but several generalizations can be made. One
is that many of the apparent discrepancies are explained by the
surprisingly difficult problem of devising markers for donor cells
that are sensitive enough and that remain stable after the cells
engraft into diverse tissues. Another is that the level of engraft-
ment of marrow-derived cells into nonhematopoietic tissues is
low unless the recipient animal is a rapidly growing embryo or
newborn animal or has injury to some organ or tissue. Unfor-
tunately, it has been extremely difficult to define the kind and
degree of tissue injury that promotes engraftment. Still another
generalization is that many of the apparent discrepancies can be
explained by subtle but critical differences in the donor cells used
for the experiments. For example, the inbred strain of mice used
for donor cells (41) or even the cell cycle of the infused cells (42)
can critically affect the results. Finally, the observations indicat-
ing that cell fusion is unexpectedly common in experiments with
adult stem cells raise a new series of unanswered questions as to
the fate of such cells during repair of tissue damage. Cell fusion
cannot explain the ability of clonally derived marrow cells to
differentiate into multiple cell phenotypes in culture (for exam-
ples, see refs. 43 and 44~. The possible contribution of cell fusion
to the engraftment and differentiation of marrow-derived cells
observed in in viva experiments has not been defined. The
available data do not resolve questions such as: Are the fused
cells dead-end products that disappear with time, or are they
intermediates in the normal process of tissue repair?
Reparative Cells from Bone Marrow: Data from Patients with
Marrow or Organ Transplants
In view of the questions raised by some of the observations in
experimental animals, it is surprising that the most convincing
evidence for Cohnheim's hypothesis appears to come from
assays on tissues of patients who have received either bone
marrow transplants or organ transplants. In female patients who
received bone marrow transplants from male donors, male cells
were found in the liver as hepatocytes and cholangiocytes (45),
in kidney as tubular epithelial cells (46), in lung as epithelial and
endothelial cells (47), in heart as cardiomyocytes (48), and in
brain as neurons (49) and Purkinje cells (50~. In most of the
reports, the number of differentiated cells detected was small. In
patients who received allografts of organ transplants, the number
of differentiated recipient cells detected in the organ was low in
many patients, but high in some instances in which pathological
changes occurred in the organ. A value of 38% of cholangiocytes
and 43% of hepatocytes were male in a male who received a
female liver and then developed fibrosing cholestatic recurrent
hepatitis C (45~. In males who received cardiac transplants from
female donors, the Y chromosome was detected by cardiac
biopsies in a mean of 0.04% of cardiomyocytes in one study (51)
and 0.16% in a second study (52~. However, 29% of the
cardiomyocytes contained the Y chromosome in "hot spots" of
pathological changes in the heart of the only 1 of the 5 patients
in the first study who died of cardiac rejection (514. In patients
who received lung allografts (53), recipient-derived cells were Mature
detected as bronchial epithelial cells, type II pneumocytes, and MS(:
seromucous glands lying adjacent to large bronchi in the trans-
planted lungs from all seven patients. The chimerism was as high
as 24% in epithelial structures displaying chronic injury. Taken
as a whole, the results indicate that although many tissues
contain stem-like cells that can participate in tissue repair, the
resident cells in tissues are replenished by cells from the blood-
stream and the bone marrow during some forms of tissue repair.
11918 1 www.pnas.org/cgi/doi/10.1073/pnas.1834138100
Which Cells from Bone Marrow?
A more difficult question to resolve has been: Which cells from
bone marrow repair injured tissues? A number of candidate cell
types are currently being explored, including marrow cells that
can serve as hematopoietic precursors (23-25, 43~. One class of
cells that has received attention for many years are the cells
currently referred to as mesenchymal stem cells or marrow
stromal cells (MSCs).
MSCs were discovered by Friedenstein and his associates (54,
55) >30 years ago. They demonstrated that a small fraction of
cells from bone marrow adhere to tissue culture surfaces and that
the adherent cells can be differentiated both in culture and in
vivo into osteoblasts, chondrocytes, and adipocytes. Friedenstein
et al.'s observations were confirmed by a large number of
subsequent investigators (4, 56-64), who demonstrated that the
cells can also differentiate in culture into muscle (65), early
precursors of neural cells (66, 67), and cardiomyocytes (683. In
parallel experiments, adherent cells from bone marrow with
most of the properties of MSCs were shown to provide effective
feeder layers for the expansion of hematopoietic stem cells (see
refs. 69 and 704. The cells have attracted considerable attention
in efforts to develop cell and gene therapies (4, 14, 18, 71),
because they are readily obtained from the patient to be treated.
Therefore their use can avoid any immune responses. Also,
extensive experiments over several decades have not shown any
evidence of the tumorgenicity that is prominently observed with
embryonic stem cells. Promising results have been reported with
use of MSCs or closely related cells from bone marrow in animal
models for a rlumber of diseases, including osteogenesis imper-
fecta (14), parkinsonism (72, 73), spinal cord injury (74-77),
stroke (78, 79), myelin deficiency (80), cardiac disorders (81-84),
and lung diseases (85, 86~. Also, several clinical trials have been
initiated and encouraging results have been reported in using
administration of MSCs for osteogenesis imperfecta (87, 88) and
Hurler's syndrome and metachromatic leukodystrophy (89) and
to enhance engraftment of heterologous bone marrow trans-
plants (904.
Properties of MSCs
Despite the great interest in MSCs, the cells are still poorly
characterized. Part of the difficulty lies in the subtle changes the
cells undergo as they are expanded in culture. The adherent cells
initially isolated from whole bone marrow readily can be cloned
as single-cell-derived colonies if they are plated at very low
densities of 1-10 cells per cm2 (43, 62, 91-934. The single-cell-
derived colonies can be differentiated into either osteoblasts,
Pre-RS cells (?)
--slowly replicating
--long telomeres
43 RS cells
--rapidly serf-renewing
--"transitory amplifying"
/// ~ '\\
Feeder
Layer
Osteo- Adipo- Chondro- Epi- Neural Other
Blasts cyte cyte thelial precursors (myo-,
endo-,
hepato-)
Fig. 1. Schematic summarizing relationship between subpopulations of
MSCs and their differentiation to specific cell phenotypes.
Prockop et a/.
OCR for page 103
Boo
Boom
4. 400
a
y 300
~0
100
. , ,
cell~fem' A
1
A
10
, _ _ ~~ 1000
r _ _
· _ _ _
0 1 2 3 ~ ~ 6 7 e ~ so 11 Always
Fig. 2. Lag period and rapid expansion of early-passage human MSCs plated
at low densities. [Reproduced with permission from ref. 93 (Copyright 2002,
Al pha Med Press).]
adipocytes, or chondrocytes in culture or in viva. Therefore, they
are multipotential and can differentiate without cell fusion.
However, the cells within the single-cell-derived colonies are
morphologically heterogeneous (57) in that they contain both
small, rapidly self-renewing cells (RS cells) and larger, more
slowly replicating cells (44, 57, 91-93~. The cultures can be
expanded rapidly and over one billion-fold in ~8 weeks. The
cultures remain relatively rich in RS cells through four or five
passages if they are maintained at low cell density. If allowed to
reach confluency, or if expanded beyond the Hayflick limit of
~50 population doublings, the cultures are dominated by larger,
more mature cells that gradually cease to proliferate (44, 63~.
Cultures enriched for RS cells have a greater potential to
t
B Heat-shocked SAE
OFP+/MSCs
Small
Airway
Epithelium
(SAE)
(X 20)
Fig. 4. Cocu Itures of G FP+ MSCs a nd heat-shocked SAECs. (A) The SAECs form
a continuous monolayer of epithelial cellsthat are broad and thin with a raised
perinuclear region. In the serum-free medium used to culture SAECs, GFP+
MSCs become thin and elongated (epifluorescence frame, Lower Right). (B)
Differential interference (Left) and epifluorescence (Right) of cocultures.
After heat shock, the monolayer of SAECs is disrupted as some of the cells
undergo necrosis and apoptosis. Over 12-120 h, some GFP+ MSCs enter the
monolayer and become broad, flat cells that reform the monolayer. (Outline
of GFP+ cell in Bottom is enhanced.) [Reproduced with permission from ref.
100 (Copyright 2003, National Academy of Sciences).]
differentiate than cultures of the large, mature cells. However,
confluent cultures of the large, mature cells continue to secrete
a number of growth factors, an observation consistent with their
ability to serve as feeder layers for hematopoietic stem cells.
By analogy with the hematopoietic stem cell system (69, 70),
the RS cells seen in early-passage, low-density cultures (Fig. 1)
are comparable to transitory amplifying cells that also replicate
rapidly in culture. Therefore, RS cells probably do not constitute
the earliest progenitors in the pathway. Also, they may not
account for all of the marrow-derived cells that are seen to
engraft and differentiate in many of the animal models that have
been studied. Some of the results obtained with experiments in
Wnt 5a (-)
Dkk-1 ( ~ )
Replating at clonal density
400
300
200
100
Cells/colony
/
1 2 3 4 5 6 7 8 9 10 11 12 13
Dad
Fig. 3. Scheme summarizing transitions of colonies of MSCs from lag period to log phase and stationary phase of growth. As indicated, Dkk-1 is synthesized
and secreted as late log/early log phase and stimulates expansion of cells. Wnt5a in expressed et the end of log phase and in stationary phase. Asthe cells expand
in the colonies, spindle-shaped RS cells give rise to larger, more mature cells. For four or five passages of preparation of MSCs the sequence is repeated after the
cells are lifted and replated at low density.
Prockop et a/.
PNAS | September 30, 2003 | vol. 100 | suppl. 1 | 11919
OCR for page 104
Epifluorescence
DIC ~ epifluorescence
Arrowheads: Taunt epithellial cells
Arrows (white and yellows: GFPi hMSCs
Arrows (red): Muli'nucleated cells formed by cell fusion
Enlargement
of last frame
Fig. 5. Time-lapse photomicroscopy of cocultures of GFP-labeled human MSCs added to heat-shocked SAECs demonstrating cell fusion. Epifluorescence and
differential interference (DIC) photomicrographs were taken every 20 min for >114 h, and selective frames for three sequences are shown. In alternate rows,
the epifluorescence and DIC frames are superimposed. [Reproduced with permission from ref. 100 (Copyright 2003, National Academy of Sciences).]
vivo may be explained by other subfractions of marrow cells. For
example, Verfaillie and her associates (24, 43) isolated marrow
cells referred to as multipotential adult progenitor cells
(MAPCs) that are more multipotential than MSCs in that they
can differentiate into hematopoietic cells. They also may more
readily differentiate into other cell phenotypes and are incor-
porated into many tissues after injection into mouse embryos.
MAPCs differ from MSCs in that they appear in cultures after
extensive incubation in medium containing low concentrations
of serum and a mixture of growth factors, express telomerase,
and are apparently immortal in culture. Another possible can-
didate is the rare hematopoietic stem cell isolated by Krause et
al. (23) that can engraft in viva as epithelial cells. The current
data, however, do not exclude a number of other possibilities as
to which cells in marrow can most effectively home to and repair
injured tissues. The most likely possibility is that some subfrac-
tions of cells more effective in repairing one type of tissue injury
but other subfractions are more effective in repairing other types
~ . . .
Of tissue Injury.
Recent Results
Conditions for Culture and Expansion of MSCs. If marrow cells are to
be used for cell and gene therapy, it will be important to define
the conditions for isolation and expansion of the cells. As
demonstrated by Friedenstein and colleagues (54, 55), MSCs are
relatively easy to isolate from marrow from most species by their
adherence to tissue culture plates and flasks. However, the cells
display several unusual features as they expand in culture. One
11920 1 www.pnas.org/cgi/doi/10.1073/pnas.1834138100
of the intriguing observations (63, 91) made with early-passage
MSCs is that they display a prolonged lag phase of 3-4 days
whenever they are plated at low density in tissue culture (Fig. 2).
The lag phase is followed by a phase of rapid exponential growth
during which the cells have doubling times as short as 10 h (93).
The cultures then pass into a stationary phase in which single-
cell-derived colonies markedly decease their rate of proliferation
without colony-to-colony contact (44, 63, 91). If the stationary-
phase cultures are replated at low density, they replicate the lag
phase, exponential growth phase, and stationary phase through
four or five passages. In initial experiments, we observed that
conditioned medium from stationary cultures increased expan-
sion of MSCs when added to newly initiated cultures (94). One
of the active components in the conditioned medium had a
molecular mass of ~30 kDa. Recently, we (95) demonstrated
that the active principal in the conditioned medium was Dick-
kopf-1 (Dkk-1), an inhibitor of the canonical Wnt signaling
pathway. The addition of recombinant Dkk-1 decreased the lag
period (Fig. 2), and antibodies to Dkk-1 prolonged the lag
period. In the early log phase, MSCs synthesized and secreted
considerable quantities of Dkk-1, but expression of the Dkk-1
gene and its receptor LRP6 ceased as the cells approached the
stationary phase. As the cultures expanded, the gene for WntSa
demonstrated a reverse pattern of expression in that the gene
was not expressed in early log-phase cultures, but was expressed
in stationary cultures (Fig. 34. The expression of WntSa by
stationary-phase cultures was consistent with their role as feeder
layers for hematopoietic stem cells, because recent observations
Prockop et a/.
OCR for page 105
.
Fig. 6. Evidence for nuclear fusion in cocultures of GFP-labeled male MSCs
and heat-shocked female SAECs. After coculture, fluorescence-activated cell
sorting was used to isolate cells positive both for GFP and CD24, a surface
epitope expressed on the epithelial cells but not on MSCs. The isolated cells
were then assayed by in situ hybridization for the X and Y chromosome. (A)
Control male cell (arrows) containing one X and one Y chromosome. (B) A rare
cell with a single nucleus containing one Y chromosome and five X chromo-
somes. (C) A highlighted cell with a single nucleus containing one Y and
three X chromosomes. (Inset) Enlargement to demonstrate the three X chro-
mosomes. The second nucleus has one X and one Y chromosome, an obser-
vation consistent with differentiation without cell fusion of a male MSC.
indicate that several Wnt ligands enhance hematopoiesis
(96-99~.
A Simplified System for Studying Repair by MSCs in Culture. The
difficulty of carrying out experiments in animal models with
MSCs and other marrow cells has prompted us to develop a
coculture system to study the repair of injured cells and tissues
by MSCs. In initial experiments, we (100) cocultured MSCs with
heat-shocked human small airway epithelial cells (SAECs).
In culture, SAECs formed a continuous monolayer of broad,
flat cells with a raised perinuclear region (Fig. 44. In the same
serum-free medium used to culture SAECs, MSCs became thin
and elongated. Therefore, as a first approximation, it was
possible to follow differentiation of the MSCs by light micros-
copy. After heat shock of the SAEC cultures, the epithelial
monolayer was disrupted as many of the cells became necrotic
and apoptotic. After addition of MSCs labeled with GFP to the
heat-shocked SAECs, some of the GFP+ MSCs entered
the monolayer and assumed the broad, flat morphology of the
epithelial cells. Assays by immunohistochemistry and cDNA
microarrays demonstrated that the GFP+ MSCs began to express
many proteins characteristic of epithelial cells and they formed
adherers junctions (data not shown). Time-lapse photomicros-
copy (Fig. 5) demonstrated that many of the GFP+ MSCs
assumed the characteristic broad morphology of epithelial cells
directly as they entered the monolayer (data not shown), but
some of the cells fused with epithelial cells to form multinuclear
Prockop et a/.
Fig. 7. Schematic showing that in the coculture system some MSCs differ-
entiated directly into SAECs and others fused. In addition, the MSCs probably
synthesize and secrete growth factors that enhance regeneration of epithel ial
and other cells.
cells (Fig. 53. Reverse-tracking of 381 GFP+ cells that had
assumed an epithelial morphology indicated that most of the
MSCs differentiated without any evidence of cell fusion. How-
ever, ~14% had undergone a distinct cell fusion event.
Differentiation of the MCSs was also examined by sorting the
cocultures for cells that were positive both for GFP and CD24,
an epitope found on the epithelial cells but not on the MSCs.
After coculture for 1 wk. assays by flow cytometry demonstrated
that ~4% of the added GFP+ cells were positive for both GFP
and CD24. About three-quarters of the isolated doubly positive
cells were mononucleated. One-quarter were binucleated, an
observation consistent with cell fusion.
In further experiments, male GFP+ MSCs were cocultured
with female heat-shocked SAECS, GFP+/CD24+ cells were
isolated from the cocultures, and the double-labeled cells were
assayed for the X and Y chromosome by in situ hybridization
(Fig. 6~. GFP+/CD24+ cells were seen that contained a single
nucleus with one Y and one X chromosome, an observation
consistent with direct differentiation of a male MSC. A number
of cells contained a single nucleus with one Y and three X
chromosomes, indicating that a male MSC and a female SAEC
had undergone both cell fusion and nuclear fusion. A rare cell
was found with a single nucleus that contained one Y and five
X chromosomes, indicating fusion of three nuclei, one from a
male GFP+ MSC cell, and two from female SAECs.
Conclusions
The cyclical expression of Dkk-1 and WntSa by MSCs in culture
provides a simple explanation for why the cells repeatedly display
a lag phase, an exponential growth phase, and a stationary phase
as they are passed in culture (63, 914. Within bone marrow, the
expression of Wnt5a instead of Dkk-1 may prevent MSCs from
expanding at the extremely rapid rate seen in low-density
cultures (Fig. 1) and thereby prevent them from overpopulating
the marrow. Also, the expression of WntSa is consistent with
effectiveness of MSCs in serving as feeder layers for hemato-
poietic stem cells, because recent observations indicate that
several Wnt ligands enhance hematopoieses (96-994. The results
also raise the possibility that recombinant Dkk-1 or analogues
can be used to enhance the expansion of RS cells in culture and
perhaps in vivo. In related experiments (95), we observed that
Dkk-1 was secreted by two lines of osteosarcoma cells as they
exited a lag phase similar to the lag phase seen with MSCs.
PNAS | September 30, 2003 I vol. 100 I supp~. ~ | 11921
OCR for page 106
Therefore, it is possible that antagonists of Dkk-1 may be useful
to limit the growth of some malignancies.
The coculture system with MSCs and epithelial cells made it
possible to demonstrate directly the response of MSCs to cell
injury (100~. Up to 4% of the added MSCs differentiated into
epithelial cells. Therefore it should be possible to use the system
to isolate the signals released by the heat-shocked epithelial cells
that attract MSCs and initiate their differentiation. Most of the
MSCs that differentiated did so without any evidence of a cell
fusion event. However, cell fusion occurred with up to one-
quarter of the cells that acquired an epithelial phenotype (Fig.
7~. Fusion of the MSCs and the epithelial cells was confirmed by
the observation that some of the cells also underwent nuclear
fusion. The rate of cell fusion was unexpectedly high for a system
1. Tanaka, E. M. (2003) Cell 113, 559-562.
2. Cohnheim, J. (1867) Pathol. Anat. Physiol. Klin. Med. 40, 1-79.
3. Ross, R., Everett, N. B. & Tyler, R. (1970) J. Cell Biol. 44, 645-654.
4. Prockop, D. J. (1997) Science 276, 71-74.
5. Doherty, M. J. & Canfield, A. E. (1999) Crit. Rev. Eukaryotic Gene Expression
9, 1-17.
6. Majka, S. M., Jackson, D. A., Kienstra, K. A., Majesky, M. W., Goodell, M. A.
& Hirschi, K. K. (2003) J. Clin. Invest. 111, 29-30.
7. LaBarge, M. A. & Blau, H. M. (2002) Cell 111, 589-601.
8. Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J.,
Benhaim, P., Lorenz, H. P. & Hedrick, M. H. (2001) Tissue End 7, 211-228.
9. Campagnoli, C., Roberts, I. A., Kumar, S., Bennett, P. R., Bellantuono, I. &
Fisk, N. M. (2001) Blood 98, 2396-2402.
10. Wulf, G. G., Luo, K. L., Jackson, K. A., Brenner, M. K. & Goodell, M. A.
(2003) Haematologica 88, 368-378.
11. De Bari, C., Dell'Accio, F., Vandenabeele, F., Vermeesch, J. R., Raymackers,
J. M. & Luyten, F. P. (2003) J. Cell Biol. 160, 909-918.
12. Taupin, P. & Gage, F. H. (2002) J. Neurosci. Res. 69, 745-749.
13. Pereira, R. F., Halford, K. W., O'Hara, M. D., Leeper, D. B., Sokolov, B. P.,
Pollard, M. D., Bagasra, O. & Prockop, D. J. (1995) Proc. Natl. Acad. Sci. USA
92, 4857-4861.
14. Pereira, R. F., O'Hara, M. D., Laptev, A. V., Halford, K. W., Pollard, M. D.,
Class, R., Simon, D., Livezey, K. & Prockop, D. J. (1998) Proc. Natl. Acad. Sci.
USA 95, 1142-1147.
15. Eglitis, M. A. & Mezey, E. (1997) Proc. Natl. Acad. Sci. USA 94, 4080-4085.
16. Brazelton, T. R., Rossi, F. M., Keshet, G. I. & Blau, H. M. (2000) Science 290,
1672-1674.
17. Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A.,
Cossu, G. & Mavilio, F. (1998) Science 279, 1528-1530.
18. Azizi, S. A., Stokes, D., Augelli, B. J., DiGirolamo, C. & Prockop, D. J. (1998)
Proc. Natl. Acad. Sci. USA 95, 3908-3913.
19. Kopen, G. C., Prockop, D. J. & Phinney, D. G. (1999) Proc. Natl. Acad. Sci.
USA 96, 10711-10716.
20. Hou, Z., Nguyen, Q., Frenkel, B., Nilsson, S. K., Milne, M., van Wijnen, A. J.,
Stein, J. L., Quesenberry, P., Lian, J. B. & Stein, G. S. (1999) Proc. Natl. A cad.
Sci. USA 96, 7294-7299.
21. Eglitis, M. A., Dawson, D., Park, K. W. & Mouradian, M. M. (1999)
NeuroReport 10, 1289-1292.
22. Liechty, K. W., MacKenzie, T. C., Shaaban, A. F., Radu, A., Moseley, A. M.,
Deans, R., Marshak, D. R. & Flake, A. W. (2000) Nat. Med. 6, 1282-1286.
23. Krause, D. S., Thiese, N. D., Collector, M. I., Henegariu, O., Hwang, S.,
Gardner, R., Neutzel, S. & Sharkis, S. J. (2001) Cell 105, 369-377.
24. Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E, Keene, C. D.,
Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., et al.
(2002) Nature 418, 41-49.
25. Kale, S., Karihaloo, A., Clark, P. R., Kashgarian, M., Krause, D. S. & Cantley,
L. G. (2003) J. Clin. Invest. 112, 42-49.
26. Orlic, D. (2002) Int. J. Hematol. 76, 144-145.
27. Badiavas, E. V., Abedi, M., Butmarc, J., Galanga, V. & Quesenberry, P. (2003)
J. Cell Physiol. 196, 245-250.
28. Chesney, J. & Bucala, R. (2000) Curr. Rheumatol. Rep. 2, 501-505.
29. Kuznetsov, S. A., Mankani, M. H., Gronthos, S., Satomura, K., Bianco, P. &
Robey, P. G. (2001) J. Cell Biol. 153, 1133-1 140.
30. Zvaifler, N. J., Marinova-Mutafchieva, L., Adams, G., Edwards, C. J., Moss,
J., Burger, J. A. & Maini, R. N. (2000) Arthritis Res. 2, 477-488.
31. Wagers, A. J., Sherwood, R. I., Christensen, J. L. & Weissman, I. L. (2002)
Science 297, 2256-2259.
32. Theise, N. D., Krause, D. S. & Sharkis, S. (2003) Science 299, 1317a.
33. Wagers, A. J., Sherwood, R. I., Christensen, J. L. & Weissman, I. L. (2003)
Science 299, 1317b.
11922 1 www.pnas.org/cgi/doi/10.1073/pnas.1834138100
in which the process was not driven by membrane damage or
amplified by selective pressure (see refs. 37-39~. At the moment
it is not clear whether the high rate of cell fusion is a urtique
property of MSCs or some fortuitous combination of the MSCs
and the heat-shocked epithelial cells. Also, because proliferation
of cells in the system ceased after ~48 h under the conditions
used here, it was not possible to define the fate of the fused cells.
Further refinement of the coculture system should make it
possible to resolve these and other issues.
This work was supported by National Institutes of Health Grants
AR47796 and AR44210, the Oberkotter Foundation, the Louisiana
Gene Therapy Research Consortium, and HCA, The Healthcare
Company.
59
60
~1
34. CastrO, R. F., Jackson, K. A., Goodell, M. A., Robertson, C. S., Liu, H. &
Shine, H. D. (2002) Science 297, 1299.
35. Blau, H., Brazelton, T., Keshet, G. & Rossi, F. (2002) Science 298, 361-362.
36. Castro, F. F., Jackson, K. A., Goodell, M. A., Robertson, C. S., Liu, H. &
Shine, H. D. (2002) Science 298, 362.
37. Ying, Q.-L., Nichols, J., Evans, E. P. & Smith, A. G. (2002) Nature 416,
545-547.
38. Terada, N., Hamazaki, T., Oka, M., Hoki, M., Mastalerz, D. M., Nakano, Y.,
Meyer, E. M., Morel, L., Petersen, B. E. & Scott, E. W. (2002) Nature 416,
542-545.
39. Wang, X., Willenbring, H., Akkari, Y., Torimaru, Y., Foster, M., Al-Dhalimy,
M., Lagasse, E., Finegold, M., Olson, S. & Grompe, M. (2003) Nature 422,
897-901.
40. Vassilopoulos, G., Wang, P. R. & Russell, D. W. (2003) Nature 422, 901-904.
41. Phinney, D. G., Kopen, G., Isaacson, R. L. & Prockop, D. J. (1999) J. Cell
Biochem. 72, 570-585.
42. Lambert, J. F., Liu, M., Colvin, G. A., Dooner, M., McAuliffe, C. I., Becker,
P. S., Forget, B. G., Weissman, S. M. & Quesenberry, P. J. (2003) J. Exp. Med.
197, 1563-1572.
43. Reyes, M., Lund, T., Lenvik, T., Aguiar, D., Koodie, L. & Verfaillie, C. M.
(2001) Blood 98, 2615-2625.
44. DiGirolamo, C. M., Stokes, D., Colter, D., Phinney, D. G., Class, R. &
Prockop, D. J. (1999) Br. J. Haematol. 107, 275-281.
45. Theise, N. D., Nimmakayalu, M., Gardner, R., Illei, P. B., Morgan, G.,
Teperman, L., Henegar, O. & Krause, D. S. (2000) Hepatology 32, 11-16.
46. Poulsom, R., Forbes, S. J., Hodivala-Dilke, K., Ryan, E., Wyles, S., Navarat-
narasah, S., Jeffery, R., Hunt, T., Alison, M., Cook, T., et al. (2001 ) J. Pathol.
195, 229-235.
47. Suratt, B. T., Cool, C. D., Serls, A. E., Chen, I., Varella-Garcia, M., Shpall,
E. J., Brown, K. K. & Worthen, G. S. (2003) Am. J. Respir. Crit. Care Med. 168,
318-322.
48. Deb, A., Wang, S., Skelding, K. A., Miller, D., Simper, D. & Caplice, N. M.
(2003) Circulation 107, 1247-1249.
49. Mezey, E., Key, S., Vogelsang, G., Szalayova, I., Lange, G. D. & Crain, B.
(2003) Proc. Natl. Acad. Sci. USA 100, 1364-1369.
50. Weimann, J. M., Charlton, C. A., Brazelton, T. R., Hackman, R. C. & Blau,
H. M. (2003) Proc. Natl. Acad. Sci. USA 100, 2088-2093.
51. Laflamme, M. A., Myerson, D., Saffitz, J. E. & Murry, C. E. (2002) Circ. Res.
90, 634-640.
52. Muller, P., Pfeiffer, P., Koglin, J., Schafers, H. J., Seeland, U., Janzen, I.,
Urbschat, S. & Bohm, M. (2002) Circulation 106, 31-35.
53. Kleeberger, W., Versmold, A., Rothamel, T., Glockner, S., Bredt, M.,
Haverich, A., Lehman, U. & Kreipe, H. (2003)Am. J. Pathol. 162, 1487-1494.
54. Friedenstein, A. J., Gorskaja, J. F. & Kulagina, N. N. (1976) Exp. Hematol. 4,
267-274.
55. Friedenstein, A. J., Chailakhyan, R. K. & Gerasimov, U. V. (1987) Cell Tissue
Kinet. 20, 263-272.
56. Castro-Malaspina, H., Gay, R. E., Resnick, G., Kapoor, N., Meyers, P.,
Chiarieri, D., McKenzie, S., Broxmeyer, H. E. & Moore, M. A. (1980) Blood
56, 289-301.
57. Mets, T. & Verdonk, G. (1981) Mech. Ageing Dev. 16, 81-89.
58. Piersma, A. H., Brockbank, K. G., Ploemacher, R. E., van Vliet, E., Brakel-van
Peer, K. M. & Visser, P. J. (1985) Exp. Hematol. 13, 237-243.
,. Owen, M. & Friedenstein, A. J. (1988) Ciba Found. Symp. 136, 42-60.
Caplan. A. I. (1991) J. Orthop. Res. 9, 641-650.
. Beresford, J. N., Bennett, J. H., Devlin, C., Leboy, P. S. & Owen, M. E. (1992)
J. Cell Sci. 102, 341-351.
62. Kuznetsov, S. A., Krebsbach, P. H., Satomura, K., Kerr, J., Riminucci, M.,
Benayahu, D. & Robey, P. G. (1997) J. Bone Miner. Res. 12, 1335-1347.
63. Bruder, S. P., Jaiswal, N. & Haynesworth, S. E. (1997) J. Cell Biochem. 64,
278-294.
Prockop et a/.
OCR for page 107
~ At" \~'
64. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R.,
Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S. & Marshak, D. R.
(1999) Science 284, 143-147.
65. Wakitani, S., Saito, T. & Caplan, A. I. (1995) Muscle Nerve 18, 1417-1426.
66. Woodbury, D., Schwarz, E. J., Prockop, D. J. & Black, I. B. (2000) J. Neurosci.
Res. 61, 364-370.
67. Sanchez-Ramos, J., Song, S., Cardozo-Pelaez, F., Hazzi, C., Stedeford, T.,
Willing, A., Freeman, T. B., Saporta, S., Janssen, W., Patel, N., et al. (2000)
Exp. Neurol. 164, 247-256.
68. Fukuda, K. (2001) Artif: Organs 25, 187-193.
69. Eaves, C., Glimm, H., Eisterer, W., Audet, J., Maguer-Satta, V. & Piret, J.
(2001 ) Ann. N.Y. Acad. Sci. 938, 63-70.
70. Wagers, A. J., Christensen, J. L. & Weissman, I. L. (2002) Gene Ther. 9'
606-612.
71. Caplan, A. I. (1990) Biomaterials 11, 44-46.
72. Schwarz, E. J., Alexander, G. M., Prockop, D. J. & Azizi, S. A. (1999) Hum.
Gene Ther. 10, 2539-2549.
73. Li, Y., Chen, J., Wang, L., Zhang, L., Lu, M. & Chopp, M. (2001) Neurosci.
Lett. 316, 67-70.
74. Chopp, M., Zhang, X. H., Li, Y., Wang, L., Chen, J., Lu, D., Lu, M. &
Rosenblum, M. (2000) NeuroReport 11, 3001-3005.
75. Hofstetter, C. P., Schwarz, E. J., Hess, D., Widenfalk, J., El Manira, A.,
Prockop, D. J. & Olson, L. (2002) Proc. Natl. Acad. Sci. USA 99, 2199-2204.
76. Akiyama, Y., Radtke, C. & Kocsis, J. D. (2002) J. Neurosci. 22, 6623-6630.
77. Wu, S., Suzuki, Y., Ejiri, Y., Noda, T., Bai, H., Kitada, M., Kataoka, K., Ohta,
M.. Chou. H. & Ide. C. (2003) J. Neurosci. Res. 72, 343-351.
78. Li, Y., Chen, J., Chen, X. G., Wang, L., Gautam, S. C., Xu, Y. X., Katakowski,
M., Zhang, L. J., Lu, M., Janakiraman, N. & Chopp, M. (2002) Neurology 59,
5 14-523.
79. Kang,S.K.,Lee,D.H.,Bae,Y.C.,Kim,H.K.,Baik,S.Y.&Jung,J.S.(2003)
Exp. Neurol., in press.
80. Jin, H. K., Carter, J. E., Huntley, G. W. & Schuchman, E. H. (2002) J. Clin.
Invest. 109, 1183-1191.
81. Wang, J. S., Shum-Tim, D., Galipeau, J., Chedrawy, E., Eliopoulos, N. & Chiu,
R. C. (2000) J. Thorac. Cardiovasc. Surg. 120, 999-1005.
82. Toma, C., Pittenger, M. F., Cahill, K. S., Byrne, B. J. & Kessler, P. D. (2002)
Circulation 105, 93-98.
83. Tomita, S., Mickle, D. A., Weisel, R. D., Jia, Z. Q., Tumiati, L. C., Allidina,
Y., Liu, P. & Li, R. K. (2002) J. Thorac. Cardiovasc. Surg. 123, 1132-1140.
Prockop et al.
84. Shake, J. G., Gruber, P. J., Baumgartner, W. A., Senechal, G., Meyers, J.,
Redmond, J. M., Pittenger, M. F. & Martin, B. J. (2002) Ann. Thorac. Surg.
73, 1919-1925.
85. Kotton, D. N., Ma, B. Y., Cardoso, W. V., Sanderson, E. A., Summer, R. S.,
Williams, M. C. & Fine, A. (2001) Development (Cambridge, U.K) 128,
5181-5188.
86. Ortiz, L. A., Gambelli, F., McBride, C., Gaupp, D., Baddoo, M., Kaminski, N.
& Phinney, D. G. (2003) Proc. Natl. Acad. Sci. USA 100, 8407-8411.
87. Horwitz, E. M., Prockop, D. J., Gordon, P. L., Koo, W. W., Fitzpatrick, L. A.,
Neel, M. D., McCarville, M. E., Orchard, P. J., Pyeritz, R. E. & Brenner, M. K.
(2001) Blood 97, 1227-1231.
88. Horwitz, E. M., Gordon, P. L., Koo, W. K., Marx, J. C., Neel, M. D., McNall,
R. Y., Muul, L. & Hofmann, T. (2002) Proc. Natl. A cad. Sci. USA 99,
8932-8937.
89. Koc, O. N., Day, J., Nieder, M., Gerson, S. L., Lazarus, H. M. & Krivit, W.
(2002) Bone Marrow Transplant. 30, 215-222.
90. Koc, O. N., Gerson, S. L., Cooper, B. W., Dyhouse, S. M., Haynesworth, S. E.,
Caplan, A. I. & Lazarus, H. M. (2000) J. Clin. Oncol. 18, 307-316.
91. Colter, D. C., Class, R., DiGirolamo, C. M. & Prockop, D. J. (2000) Proc. Natl.
Acad. Sci. USA 97, 3213-3218.
92. Colter, D. C., Sekiya, I. & Prockop, D. J. (2001) Proc. Natl. Acad. Sci. USA
98, 7841-7845.
93. Sekiya, I., Larson, B. L., Smith, J. R., Pochampally, R., Cui, J. G. & Prockop,
D. J. (2002) Stem Cells 20, 530-541.
94. Colter, D. C. (2001) Ph.D. thesis (Tulane univ., New Orleans).
95. Gregory, C. A., Singh, H., Perry, A. S. & Prockop, D. J. (2003) J. Biol. Chen~.
278, 28067-28078.
96. Austin, T. W., Solar, G. P., Ziegler, F. C., Liem, L. & Matthews, W. (1997)
Blood 89, 3624-3635.
97. Van Den Berg, D. J., Sharma, A. K., Bruno, E. & Hoffman, R. (1999) Blood
98, 3189-3202.
98. Murdoch, B., Chadwick, K., Martin, M., Shojaei, F., Shah, K. V., Gallacher,
L., Moon, R. T. & Bhatia, M. (2003) Proc. Natl. Acad. Sci. USA 100,
3422-3427.
99. Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K.,
Hintz, I., Nusse, R. & Weissman, I. L. (2003) Nature 423, 409-414.
100. Spees, J. L., Olson, S. D., Ylostalo, J., Lynch, P. J., Smith, J., Perry, A., Peister,
A., Wang, M. Y. & Prockop, D. J. (2003) Proc. Natl. Acad. Sc~. USA 100,
2397-2402.
PNA5 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11923
Representative terms from entire chapter:
cell fusion
