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This article was retracted on March 5, 2009.
PLENARY PAPER
From the Stem Cell Institute, Department of Medicine,
and Cancer Center, University of Minnesota Medical School, Minneapolis.
It is here reported that mesenchymal stem cells
known to give rise to limb-bud mesoderm can, at the single-cell level,
also differentiate into cells of visceral mesoderm and can be expanded extensively by means of clinically applicable methods. These cells were
named mesodermal progenitor cells (MPCs). MPCs were selected by
depleting bone marrow mononuclear cells from more than 30 healthy human
donors of CD45+/glycophorin-A (GlyA)+ cells.
Cells were cultured on fibronectin with epidermal growth factor and
platelet-derived growth factor BB and 2% or less fetal calf serum. It
was found that 1/5 × 103
CD45 Embryonic stem (ES) cells are pluripotent cells
derived from blastocysts that can be propagated indefinitely
undifferentiated in vitro, can differentiate to all cell lineages in
vivo, and can be induced to differentiate to most cell types in
vitro.1-4 Although ES cells have been isolated from
humans,2,3 their use in research as well as therapeutics
is encumbered by ethical considerations.5 The ability to
purify, culture, and manipulate multipotent stem cells from
nonembryonic origin would provide investigators with an invaluable cell
source to study cell and organ development. In addition, such cells
could serve to develop replacement tissues for congenital or
degenerative disorders. Stem cells have been identified in most organ
tissues, including hematopoietic,6 neural,7
gastrointestinal,8 epidermal,9 hepatic,10 and mesenchymal stem cells
(MSCs).11-14
MSCs were first identified by Fridenshtein,11 who
demonstrated that when bone marrow (BM) is plated in fetal calf serum (FCS)-containing medium, colonies of adherent fibroblastlike cells develop that differentiate into bone and adipocytes. Since then, several investigators have shown that these cells can also
differentiate into chondrocytes, adipocytes, and, at least in rodents,
skeletal myocytes.13-15 MSCs can be purified on the basis
of their ability to adhere to plastic or with monoclonal antibodies
(SH2 and SH4, or Stro1).13,14 Here, we describe for the
first time the isolation and ex vivo expansion of cells from postnatal
BM that can differentiate at the single-cell level not only into MSCs,
but also into cells of visceral mesodermal origin, such as endothelium,
which we termed mesodermal progenitor cells (MPCs).
Bone marrow
Cytokines
Antibodies Antibodies against fast-twitch skeletal myosin, sarcomeric actin, -actin, fibroblast surface antigen (1B10), and control mouse
immunoglobulin G (IgG) were from Sigma. Antibodies against Tie, Tek,
Flt1, fms-related tyrosine kinase gene 1 (Flk1), type II
collagen, type I collagen, peroxisome proliferator activated receptor
(PPAR)- , von Willebrand factor (vWF), MYO-D, and myogenin were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against
CD13, CD31, CD34, CD36, CD38, CD44, CD49b, CD49e, CD50, CDw90, CD117,
HLA-DR, 2-microglobulin, and class I HLA were from Becton Dickinson
(Mountain View, CA). Antibodies against osteonectin (LF-7), osteopontin
(BON-1), and bone sialoprotein (LF-100) were a gift from Dr L. Fisher,
National Institutes of Health (Bethesda, MD). H1P12 was a gift from Dr
R. Hebbel, University of Minnesota. Secondary goat anti-mouse or goat
anti-rabbit antibodies were from Dako (Carpinteria, CA).
Cell lines AFT024, a fetal liver myofibroblast feeder, kindly provided by Dr I. Lemishka, Princeton University, was maintained and subcultured as described.16 The HT85 human osteosarcoma cell line was obtained from ATCC (Manassas, VA). The cell line was maintained in high-glucose Dulbecco modified essential medium (DMEM; Gibco BRL, Grand Island, NY) with 10% FCS (Hyclone Laboratories, Logan, UT) until use.MPC and differentiation culture conditions MPC cultures.
We plated 5 to 10 × 103
CD45 Limiting dilutions.
To assess MPC frequency, we plated CD45 Differentiation cultures. To induce differentiation, we used MPC expansion medium without serum, EGF, and PDGF-BB unless otherwise indicated, supplemented with factors as indicated. Western blotting Protein lysates, obtained from MPCs or differentiated progeny as previously described,18 were separated on precast 7.5% to 10% polyacrylamide gel electrophoresis gels (Bio-Rad, Hercules, CA). After blocking, blots were incubated with rabbit polyclonal antibodies against Tek, type II collagen,-actin at 1:200, goat polyclonal antibodies against PPAR-, vWF at 1:100, and mouse monoclonal antibodies against fast-twitch skeletal myosin at 1:2000, washed and incubated with a goat antimouse or antirabbit horseradish peroxidase-conjugated antibody (1:10 000 dilution) (E. I. du Pont de
Nemours, Boston, MA). Bands were visualized by means of an electrogenerated chemiluminescence (ECL) detection system.
Immunohistochemistry and immunofluorescence Immunohistochemistry. Proteins were visualized by means of the labeled streptavidin biotin (LSAB+) immunoperoxidase system (Dako). Cells were fixed with 4% paraformaldehyde at 20°C, and endogenous peroxidase activity was quenched with 3% peroxide for 5 minutes. Slides were incubated sequentially for 30 minutes each with rabbit polyclonal antibodies against LF-7, BON-1, and LF-100 (1:400); rabbit polyclonal antibodies against type I and II collagen (1:50); and rabbit polyclonal antibodies against fast-twitch myosin (1:50), followed by biotin-labeled anti-rabbit or anti-goat IgG antibody and streptavidin and then for 10 minutes with 3-amino-9-ethylcarbazole. For Von Kossa staining, cells were fixed with methanol at 20°C for
2 minutes, treated with 2% silver nitrate (Sigma) in a clear glass
coplin jar placed directly in front of a 60-W lamp for 1 hour. Slides
were rinsed in dH2O, fixed with 2.5% sodium thiosulphate
(Sigma) for 5 minutes, and washed in dH2O. Cells were
counterstained with 1% neutral red (Sigma) for 1 minute and rinsed in
tap water.
For toluidine blue staining, cells were fixed with methanol at 20°C
for 2 minutes and stained in 1% toluidine blue (Sigma) in 50%
isopropanol at 37°C for 30 minutes. Slides were dehydrated in 100%
isopropanol and mounted.
For oil-red staining, cells were fixed with methanol at 20°C for 2 minutes and rinsed in 50% alcohol. Slides were stained in oil-red-O
(Sigma) for 10 minutes and rinsed in 50% alcohol. After rinsing in
dH2O, slides were counterstained with Mayer hematoxylin (Sigma) for 1 minute.
For alkaline phosphatase staining, cells were fixed with methanol at
20°C for 2 minutes and washed in 100 mM Tris-HCl, pH 9.5, 100 mM
NaCl, and 10 mM MgCl2 buffer (all from Sigma) for 10 minutes. Slides were then stained with fast 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium alkaline phosphatase substrate (Sigma) for 5 to 10 minutes and rinsed in dH2O.
Immunofluorescence.
For staining of cytoskeletal proteins, cells were fixed with methanol
at Fluorescence-activated cell sorting.
For fluorescence-activated cell sorting (FACS), cells were detached and
stained sequentially with primary antibodies and immunofluorescent secondary antibodies and fixed with 2% paraformaldehyde until analysis
with a FACSCalibur (Becton Dickinson). For detection of intracellular
proteins, cells were permeabilized with methanol/PBS for 2 minutes
at Hematopoietic assays MPCs were plated at 20 000/cm2, treated with 10 U IL-1 for 24 hours in expansion medium, and maintained afterward in Iscoves modified Dulbecco medium (IMDM) (Gibco BRL) plus 12.5% FCS plus 12.5% horse serum and 10 7 M hydrocortisone.
MPC-derived stroma and confluent AFT024 cells were irradiated at 2 Gy
as described.16 Cord blood CD34+ cells,
purified as previously described,19 were plated in contact with irradiated MPC-derived stroma or AFT024 feeders in IMDM plus 12.5% FCS plus 12.5% horse serum (Hyclone) and 107 M
hydrocortisone (Sigma) for 2 weeks. After 2 weeks, cells were harvested
and plated in clonogenic methylcellulose medium, and colony-forming
cells (CFCs) were enumerated.16
Karyotyping Cells, subcultured at a 1:4 dilution 12 hours before harvesting, were collected with trypsin-EDTA and subjected to a 1.5-hour colcemid incubation followed by lysis with hypotonic KCL and fixation in acid/alcohol as previously described.20 Metaphases were analyzed after QFQ or GTG banding.20Telomere length measurements Telomere length was measured by means of the Telomere Length Assay Kit from Pharmingen (Franklin Lakes, NJ) according to the manufacturer's recommendations.Transduction Retroviral supernatant was produced by incubating a recombinant variant of Moloney murine leukemia virus (MFG) and enhanced green fluorescent protein (eGFP) containing PG13 cells (provided by Dr G. Wagemaker, University of Rotterdam, The Netherlands)21 with MPC expansion medium for 48 hours, filtered and frozen at80°C. MPCs were incubated with retroviral supernatants and 8 µg/mL protamine (Sigma) for 6 hours. This was repeated 24 hours later. Transduction efficiency was analyzed by FACS.
Southern blot analysis for retroviral insert DNA from 0.5 to 5 × 106 MPCs or endothelial- or myoblast-differentiated progeny was prepared from cells by standard methods and digested overnight with NcoI (a single cutter in eGFP) or EcoRI (a single cutter between eGFP and the 3' long terminal repeat). Fragments were separated by gel electrophoresis, blotted to nylon, and probed with biotin-labeled eGFP-complementary DNA (cDNA) or MFG-internal ribosome entry site (IRES)-eGFP-cDNA. Hybridization of DNA digested with EcoRI with the eGFP-cDNA probe should yield a single band, whereas hybridization of DNA digested with NcoI with the MFG-IRES-eGFP-cDNA probe should yield 2 bands per retroviral insert. Detection was by ECL.
Culture of undifferentiated MPCs Our initial hypothesis was that mesodermal progenitors are present in marrow but differ from hematopoietic progenitors. We therefore selected cells that do not express hematopoietic markers (CD45 and GlyA). Following 2 depletion steps using Macs immunomagnetic beads (Miltenyi Biotec), mononuclear cells were greater than 99.5% CD45GlyA.
CD45GlyA cells constitute 0.1% to 0.5% of
BMMNCs. CD45GlyA cells were plated in wells
coated with 10 µg/mL FN with expansion medium. After 7 to 21 days,
small clusters of adherent cells developed (Figure
1A). Limiting dilution assays (n = 5)
showed that the frequency of cells giving rise to these adherent
clusters is 0.02% to 0.08% of CD45GlyA
cells. CD45+ or GlyA+ BMMNCs did not give rise
to adherent cells.
When clusters appeared (about 103 cells), cells were
replated every 3 to 5 days at a 1:4 dilution under the same culture
conditions. Cell densities were maintained between 2 and
8 × 103/cm2. Cell-doubling time was 48 to 60 hours for the initial 20 to 25 cell doublings and increased to
72 hours after 25 to 30 cell doublings. Immunophenotypic analysis by
FACS of cells obtained after 10 to 15 cell doublings (n = more than
15) showed that cells did not express CD10, CD31, CD34, CD36,
CD38, CD50, CD62E, CD106, CD117, H1P12, fibroblast surface antigen
(1B10), HLA-DR, class I HLA (Figure 2A),
CD45, Tie, or Tek (not shown). Cells expressed low levels of
When MPCs were cultured in serum-free medium supplemented with
10 ng/mL IGF-1, cell doubling was slower (longer than 60 hours), but
more than 50 cell doublings could be obtained. As was seen for cells
cultured with 2% FCS, MPCs in serum-free cultures with IGF-1 were
small (not shown), did not express HLA-DR and class I HLA,
expressed low levels of CD44 (Figure 2C), and differentiated into all mesodermal phenotypes (see below). Thus, in contrast to the
phenotype reported for MSCs,12-14 MPCs cultured at low
density and with 2% or lower FCS are class
I-HLA Average telomere length of MPCs from 5 donors (ages 2 to 50 years)
cultured for 35 cell doublings on FN with 2% FCS, 10 ng/mL EGF, and 10 ng/mL PDGF at densities between 2 and
8 × 103/cm2 was between 11 and 15 kilobases
(kb) (Figure 3A). In 3 of the 5 donors
from whom blood mononuclear cells were available, this was
approximately 3 kb longer than the telomere length in blood lymphocytes (not shown). Telomere length of cells from one donor evaluated after 15, 35, and 45 cell doublings remained unchanged. Cytogenetic analysis of MPCs recovered after 40 cell doublings (n = 2) showed a normal karyotype (Figure
4B). Although this suggests that MPCs may
have unlimited cell expansion potential, more extensive culture to
beyond 100 cell doublings will be required to fully prove that MPCs are
not subject to the Hayflick phenomenon.22 Studies are
ongoing to determine telomerase activity in these cells.
When plated on type IV collagen or laminin instead of FN, cells
expressed CD44 and class I HLA (not shown), similar to the phenotype
described for MSCs, and proliferated poorly beyond 30 cell doublings.
This suggests that signaling through the Culture with greater than 2% FCS (Figures 1C, 2D) or culture at high density (greater than 8 × 103/cm2) (Figures 1D, 2E) yielded cells that were significantly larger and more vacuolated, and caused acquisition of high levels of CD44, HLA-DR, and class I HLA and decreased levels of CD13 and CD49b. This was associated with loss of proliferation potential beyond 30 cell doublings and loss of differentiation potential (see below). High-efficiency retroviral transduction of MPCs MPCs were subcultured at a 1:4 dilution and then subjected to 2 sequential transductions 24 hours apart with a GALV-pseudotyped MFG-eGFP vector. Transduction efficiency ranged between 30% and 70% (Figure 4). GFP expression remained relatively unchanged for at least 20 population doublings, as is shown in Figure 4. In addition, the percentage of eGFP+ cells was unchanged following cryopreservation (not shown) or following differentiation (Figure 12).MPCs cultured at low density and with 2% FCS differentiate to mesenchymal cell types and skeletal myoblasts Differentiation to tissue-specific cells required addition of induction agents or cytokines. This differs from what has been seen for ES cells that differentiate spontaneously when cells are cultured as embryoid bodies (EBs). The cytokines/inducers were chosen on the basis of what has been described for differentiation of MSCs to the osteoblast, cartilage, and adipocyte lineage13,23 and for differentiation of EB to the skeletal myoblast15 lineage and endothelium.24 Differentiation to all mesodermal lineages required that cells be replated at high confluency (greater than 2 × 104/cm2). Whether this causes exit from cell cycle, required for differentiation, or whether cell-cell contact itself or factors secreted following cell-cell contact are needed for differentiation is not known.Osteoblasts MPCs, replated at 2 × 104/cm2, were cultured in DMEM with 10 mM-glycerophosphate, 107 M
dexamethasone, and 0.2 mM ascorbic acid,13,23 with media changes every 3 to 4 days. After 14 days, more than 80% of MPCs acquired markers of osteoblasts as shown by FACS for osteopontin, bone
sialoprotein, and osteonectin (Figure
5A). We could not detect cells expressing
skeletal muscle markers (fast-twitch myosin) or endothelial markers
(vWF) (FACS analysis; data not shown). These results were confirmed by
immunohistochemistry (Figure 5B) and Western blot (Figure 5C). We also
used Von Kossa staining to demonstrate the presence of calcium
phosphate after culture conditions were changed and showed that
differentiation to osteoblasts is associated with increased alkaline
phosphatase staining (Figure 5D).
Chondroblasts To induce chondroblast differentiation, MPCs were trypsinized, and 1 × 106 MPCs were cultured in 1 mL serum-free expansion medium without EGF and PDGF-BB, but with 100 ng/mL TGF-1, in the tip
of a 15-mL conical tube to allow aggregation of the cells in micromass
suspension culture.13,23 Medium was exchanged every 3 to 4 days. Development of chondroblasts was followed by staining the
micromasses with toluidine blue, which stains the proteoglycan
extracellular matrix,13,14 and with antibodies against
type II and type I collagen.25 We found that small
aggregates of cartilage formed in the bottom of the tubes that stained
positive with toluidine blue from day 14 on (Figure
6A). Expression of type II collagen was
detected from day 5 on, and by day 21, type II collagen but not type I collagen staining could be detected throughout the micromass (Figure 6A). Results were confirmed by Western blot analysis (Figure
6B).
Adipocytes To induce adipocyte differentiation, MPCs were cultured at 2 × 104/cm2 with 10% horse serum or 100 ng/mL insulin (not shown). Cultures were maintained for 14 days with medium exchanges every 3 to 4 days. After 14 days, more than 80% of cells differentiated into lipid-laden cells that stained with oil-red (Figure 7A). Using Western blot, we also showed that cells expressed PPAR- (Figure 7B).13
Although such markers are also expressed in hepatic epithelium, the
differentiated cells did not have epithelial morphology and did not
stain for other hepatocyte-specific markers, such as albumin.
Hematopoietic supportive stroma We induced "stromal" differentiation by incubating MPCs subcultured at 2 × 104/cm2 with 10 U IL-1
in expansion medium for 24 hours, after which they were switched to
IMDM without EGF and PDGF-BB, but with 12.5% FCS, 12% horse serum,
and 107 M hydrocortisone.26 On day 7, 50%
of the medium was replaced with fresh medium. To demonstrate that these
cells support hematopoiesis, feeders were irradiated at 2 Gy, and
allogeneic cord blood CD34+ cells were plated in contact
with the feeder in IMDM with FCS, horse serum, and hydrocortisone.
After 2 weeks, progeny was replated in methylcellulose assay to
enumerate CFCs. A 3- to 5-fold expansion of CFCs was seen, which was
similar to cultures of CD34+ cells in contact with the
murine fetal liver feeder, AFT02416 (Figure
8). We did not test whether treatment of
MPCs with IL-1 is required to elucidate the hematopoietic supportive
ability of MPCs. It is also not yet known if MPCs, such as, for
example, the AFT024 feeder, will support repopulating stem
cells.19,27
Skeletal myoblasts Caplan showed that rat MSCs can be induced to differentiate into skeletal myoblastlike cells.15 We therefore tested whether human MPCs can differentiate into cells of the myoblast lineage. MPCs were plated at 2 × 104/cm2 in expansion medium containing 2% FCS, 10 ng/mL EGF, and 10 ng/mL PDGF-BB as well as 3 µM 5-azacytidine15 for 24 hours. Afterward, cells were maintained in expansion medium with 2% FCS, EGF, and PDGF-BB, but without 5-azacytidine, and medium was exchanged every 3 to 4 days. We evaluated the cultures by FACS (Figure 9A), immunohistochemistry (Figure 9B), and Western blot (Figure 9C) to detect transcription factors and skeletal proteins found during muscle development.28-30 At 4 days after induction, we detected the MYO-D and myogenin transcription factors. After 15 days, we no longer detected MYO-D or myogenin, but could detect fast-twitch myosin and sarcomeric actin. When cultures were evaluated by immunohistochemistry and FACS, we found that greater than 80% of cells expressed skeletal muscle-specific transcription factors and mature muscle proteins (Figure 9A). Cells treated with 5-azacytidine did not express osteoblast-specific markers (bone sialoprotein) or endothelial markers (vWF) (FACS analysis; not shown). When cells treated with 5-azacytidine and maintained in EGF- and PDGF-BB-containing medium for 14 days were subjected to culture conditions used for osteoblast differentiation (DMEM and-glycerophosphate, see above), cells died and no
osteoblast differentiation was seen, demonstrating that the majority of
cells had lost their multilineage potential.
Thus, we demonstrate that MPCs cultured under the conditions described here, like MSC, can differentiate into osteoblastlike, chondroblastlike, adipocytelike and stromalike cells as well as skeletal muscle-like cells. MPCs cultured under low-density conditions and with 2% FCS differentiate to visceral mesoderm-derived endothelial cells Until now, there has been no evidence that MSCs cultured under conditions initially developed by Pittenger et al14 differentiate into cells with endothelial characteristics. Because undifferentiated MPCs express low levels of the VEGF receptors KDR and FLt1 (Figure 10), we tested whether they could be induced to differentiate into endothelial cells. We replated human MPCs at 2 × 104/cm2 in serum-free expansion medium without EGF and PDGF, but with 10 ng/mL VEGF-B. We evaluated progeny for markers of endothelial differentiation. Undifferentiated MPCs do not express CD31, CD34, CD36, CD62E, CD62P, H1P12, Tie, Tek, and vWF, but express low levels of VEGF-R1, Flt1, VEGF-R2, and KDR (Figure 10). Following treatment with VEGF for 14 days, greater than 80% of cells expressed CD34, vWF, CD36, Tek (Figure 10A), CD62E, CD62P, H1P12, and Tie (not shown) and expressed much higher levels of the VEGF receptors KDR (Figure 10A) and Flt1 (not shown). Results were again confirmed by immunofluorescence analysis (Figure 10B) and Western blot (Figure 10C). Whether similar results can be obtained when other VEGF isoforms are used was not evaluated. Endothelial cells differentiated from MPCs could be maintained in culture for at least 2 months when subcultured every 3 to 5 days in the presence of 10% FCS and VEGF.
No hematopoietic differentiation from MPCs Choi et al24 have shown that Flk1-positive cells present in murine EBs can, depending on the culture conditions, be induced to differentiate into CD34+ endothelial cells as well CD34+ hematopoietic cells. Because we had shown that KDRdim MPCs could be induced to differentiate to endothelial cells, we also tested whether KDRdim MPCs could be induced to differentiate into hematopoietic cells. The eGFP-transduced MPCs were cocultured in contact with the AFT024 feeder, or in supernatants from this feeder with different combinations of cytokines, including 10 to 20 ng/mL VEGF, bFGF, BMP-4, Flt3-L, SCF, IL-3, IL-6, G-CSF, GM-CSF, thrombopoietin, and erythropoietin, for 2 to 6 weeks and were analyzed by FACS for expression of CD45 and/or CD34. Although eGFP+ CD34+ cells could be detected, they did not costain with CD45, indicating their endothelial nature. No CD45+ cells have been detected under any of the culture conditions tested. Therefore, we have been unable to induce MPCs to differentiate to CD34+/CD45+ hematopoietic cells despite culture of MPCs on the AFT024 feeder, which is derived from murine fetal liver and is known to support murine hematopoietic stem cells (HSCs) as well as human marrow-, peripheral blood-, and cord blood-derived primitive progenitors.19,27,31 Studies are ongoing to test additional feeders and cytokine combinations for their ability to induce hematopoietic differentiation from MPCs.Differentiation from MPCs cultured with 10% FCS or without FCS In contrast to differentiation to the mesenchymal cell types osteoblasts, chondroblasts, adipocytes, and stromal cells, differentiation to skeletal muscle and endothelium required that the initiating cell population be CD44 and class
I-HLA. Thus, when MPC cultures were maintained
with greater than 2% FCS (Figure 11)
or at high density (greater than 104/cm2) (not
shown), no skeletal muscle or endothelial differentiation was seen.
Like MPCs cultured with 2% FCS and EGF plus PDGF-BB, MPCs that had
been cultured in serum-free MPC medium supplemented with EGF and
PDGF-BB but also with IGF could be induced to differentiate to
osteoblasts, chondroblasts, adipocytes, and stromal cells, as well as
to skeletal myoblasts and endothelium (Figure 11).
Single-cell origin of limb-bud and visceral mesoderm progeny To prove single-cell derivation of the differentiated cell types, we initially plated more than 2000 cells recovered from established cultures as single cells in FN-coated 96-well plates in MPC expansion medium. In no well did we detect cell growth. However, when cells were deposited at 10 cells per well, progeny of 0.2% of wells could be expanded to more than 107 cells. Thus, MPCs themselves or accessory cells may secrete growth factors, cytokines, or extracellular matrix components required for their growth. This is currently under evaluation.An alternative method for proving single-cell origin of differentiated
progeny is retroviral marking.32,33 In the first experiment, MPCs obtained after 20 cell doublings were transduced with
an MFG-eGFP retrovirus, and eGFP+ cells were selected by
FACS. Cells were deposited as 10 cells per well and expanded in culture
until greater than 2 × 107 cells were obtained. Then,
5 × 106 undifferentiated MPCs were collected for
Southern blot analysis, and further groups of MPCs, consisting of
5 × 106 cells for each group, were induced to
differentiate to osteoblasts, adipocytes, stroma cells, skeletal
myoblasts, and endothelium. After 14 days under
differentiation conditions, cells were harvested and used for Southern
blot (0.5 to 2 × 106 cells) and Western blot (0.5 to
2 × 106 cells) analysis. We examined 3 independent
populations. Results for Western blot studies are shown for clones nos.
1, 2, and 3 in Figures 6, 7, 9, and 11. Results from
the Southern blot for clone no. 3 is shown in Figure
12A. A single retroviral insert was
seen in MPCs, skeletal myoblasts, endothelium, and osteoblasts. No
insert could be detected in stroma, possibly owing to the fact that DNA
from only 0.5 × 106 cells was loaded.
In the second experiment, a fraction of MPCs obtained after 20 cell doublings was transduced and eGFP+ MPCs were diluted in nontransduced MPCs from the same donor to obtain a final concentration of 5%. These mixtures were plated at 100 cells per well, and culture was expanded until greater than 2 × 107 cells were obtained. Then, 5 × 106 MPCs were collected for Southern blot analysis, and groups of 5 × 106 MPCs were each induced to differentiate to skeletal myoblasts and endothelium. After 14 days under differentiation conditions, cells were harvested and used for Southern blot analysis (0.5 to 2 × 106 cells). Cells were also fixed and subjected to immunofluorescence evaluation for fast-twitch myosin and eGFP (skeletal muscle cultures) or vWF and eGFP (endothelial cultures). We found a single retroviral insertion site in MPCs and skeletal and endothelial differentiated progeny (clone 2A3, Figure 12C). Immunofluorescence evaluation showed that 8% of cells in clone 2A3 that were induced to differentiate with 5-azacytidine stained positive for eGFP and fast-twitch myosin, and 7% of cells that were induced to differentiate to endothelium costained for eGFP and vWF (Figure 12D). These studies therefore definitively prove for the first time that mesodermal progenitors exist in human marrow and that these can differentiate at the single-cell level in cells of limb-bud and visceral mesodermal lineage. Conclusion We describe here for the first time the isolation and ex vivo culture of mesodermal progenitor cells that can be expanded beyond 50 cell doublings and can, at the single-cell level, generate both limb-bud and visceral mesodermal cell types. MPCs are CD44, class I HLA, and
2-microglobulin, characteristics of classical
mesenchymal stem cells.14 MPCs are also
CD34, CD45, and CD117 and
therefore differ from primitive hematopoietic progenitors, which have
recently also been associated with "stem cell
plasticity."34-36 However, as has been suggested in
these reports,34-36 we also have preliminary evidence that
MPCs may be capable of differentiating into nonmesodermal cell types,
such as neuroectoderm, and this at the single-cell level (manuscript in preparation).
In contrast to HSCs, we show that MPCs can be expanded ex vivo for more than 50 population doublings without obvious signs of differentiation or senescence and can be readily transduced with retroviral vectors. Provided that MPCs engraft long term, which is currently being tested, they may be a much better source of cells than HSCs for gene therapy of congenital disorders characterized by enzyme deficiencies, such as Gaucher disease,37 mucopolisaccharidosis,38 leukodystrophies,39 and others. Like MPCs, ES cells can differentiate into all mesodermal cell types and may have an even greater proliferation potential. However, MPCs but not ES cells can be derived from BM from most healthy donors irrespective of age, providing for an autologous source of stem cells. Thus, because MPCs can be selected and expanded under conditions that should be readily adaptable to production by clinical good manufacturing process and are easily transduced with retroviral vectors, they may be an ideal source of cells for therapy of degenerative or traumatic disorders of mesodermal cells or for therapy of single gene disorders.
Submitted April 9, 2001; accepted June 25, 2001.
Supported by National Institutes of Health grants PO1-CA-65493-1 (C.M.V.) and 1F31AI/GM10291 (M.R.); Children's Cancer Research Fund; and the University of Minnesota Bone Marrow Transplant Research Fund; C.M.V. is a Scholar of the Leukemia Society of America.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Catherine M. Verfaillie, Professor of Medicine, University of Minnesota, MMC 716, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: verfa001{at}umn.edu.
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 V. Kindler Postnatal stem cell survival: does the niche, a rare harbor where to resist the ebb tide of differentiation, also provide lineage-specific instructions? J. Leukoc. Biol., October 1, 2005; 78(4): 836 - 844. [Abstract] [Full Text] [PDF]
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C. Pan, M. S. Nelson, M. Reyes, L. Koodie, J. J. Brazil, E. J. Stephenson, R. C. Zhao, C. Peters, S. B. Selleck, S. E. Stringer, et al. Functional abnormalities of heparan sulfate in mucopolysaccharidosis-I are associated with defective biologic activity of FGF-2 on human multipotent progenitor cells Blood, September 15, 2005; 106(6): 1956 - 1964. [Abstract] [Full Text] [PDF]
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N. Bertani, P. Malatesta, G. Volpi, P. Sonego, and R. Perris Neurogenic potential of human mesenchymal stem cells revisited: analysis by immunostaining, time-lapse video and microarray J. Cell Sci., September 1, 2005; 118(17): 3925 - 3936. [Abstract] [Full Text] [PDF]
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T. Tondreau, N. Meuleman, A. Delforge, M. Dejeneffe, R. Leroy, M. Massy, C. Mortier, D. Bron, and L. Lagneaux Mesenchymal Stem Cells Derived from CD133-Positive Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression, and Plasticity Stem Cells, September 1, 2005; 23(8): 1105 - 1112. [Abstract] [Full Text] [PDF]
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S. M. Majka, M. A. Beutz, M. Hagen, A. A. Izzo, N. Voelkel, and K. M. Helm Identification of Novel Resident Pulmonary Stem Cells: Form and Function of the Lung Side Population Stem Cells, September 1, 2005; 23(8): 1073 - 1081. [Abstract] [Full Text] [PDF]
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V. Sordi, M. L. Malosio, F. Marchesi, A. Mercalli, R. Melzi, T. Giordano, N. Belmonte, G. Ferrari, B. E. Leone, F. Bertuzzi, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets Blood, July 15, 2005; 106(2): 419 - 427. [Abstract] [Full Text] [PDF]
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Y. Sato, H. Araki, J. Kato, K. Nakamura, Y. Kawano, M. Kobune, T. Sato, K. Miyanishi, T. Takayama, M. Takahashi, et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion Blood, July 15, 2005; 106(2): 756 - 763. [Abstract] [Full Text] [PDF]
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 Y. L. Tang, Q. Zhao, X. Qin, L. Shen, L. Cheng, J. Ge, and M. I. Phillips Paracrine Action Enhances the Effects of Autologous Mesenchymal Stem Cell Transplantation on Vascular Regeneration in Rat Model of Myocardial Infarction Ann. Thorac. Surg., July 1, 2005; 80(1): 229 - 237. [Abstract] [Full Text] [PDF]
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 B. M. Deasy, B. M. Gharaibeh, J. B. Pollett, M. M. Jones, M. A. Lucas, Y. Kanda, and J. Huard Long-Term Self-Renewal of Postnatal Muscle-derived Stem Cells Mol. Biol. Cell, July 1, 2005; 16(7): 3323 - 3333. [Abstract] [Full Text] [PDF]
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A.-M. Rodriguez, D. Pisani, C. A. Dechesne, C. Turc-Carel, J.-Y. Kurzenne, B. Wdziekonski, A. Villageois, C. Bagnis, J.-P. Breittmayer, H. Groux, et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse J. Exp. Med., May 2, 2005; 201(9): 1397 - 1405. [Abstract] [Full Text] [PDF]
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C. Moriscot, F. de Fraipont, M.-J. Richard, M. Marchand, P. Savatier, D. Bosco, M. Favrot, and P.-Y. Benhamou Human Bone Marrow Mesenchymal Stem Cells Can Express Insulin and Key Transcription Factors of the Endocrine Pancreas Developmental Pathway upon Genetic and/or Microenvironmental Manipulation In Vitro Stem Cells, April 1, 2005; 23(4): 594 - 603. [Abstract] [Full Text] [PDF]
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B. Fang, C. Zheng, L. Liao, Q. Han, Z. Sun, X. Jiang, and R. C. H. Zhao Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics Blood, April 1, 2005; 105(7): 2733 - 2740. [Abstract] [Full Text] [PDF]
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S. Glennie, I. Soeiro, P. J. Dyson, E. W.-F. Lam, and F. Dazzi Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells Blood, April 1, 2005; 105(7): 2821 - 2827. [Abstract] [Full Text] [PDF]
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G.-R. Li, H. Sun, X. Deng, and C.-P. Lau Characterization of Ionic Currents in Human Mesenchymal Stem Cells from Bone Marrow Stem Cells, March 1, 2005; 23(3): 371 - 382. [Abstract] [Full Text] [PDF]
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E. Steck, H. Bertram, R. Abel, B. Chen, A. Winter, and W. Richter Induction of Intervertebral Disc-Like Cells From Adult Mesenchymal Stem Cells Stem Cells, March 1, 2005; 23(3): 403 - 411. [Abstract] [Full Text] [PDF]
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 M. Iwasaki, Y. Adachi, K. Minamino, Y. Suzuki, Y. Zhang, M. Okigaki, K. Nakano, Y. Koike, J. Wang, H. Mukaide, et al. Mobilization of Bone Marrow Cells by G-CSF Rescues Mice from Cisplatin-Induced Renal Failure, and M-CSF Enhances the Effects of G-CSF J. Am. Soc. Nephrol., March 1, 2005; 16(3): 658 - 666. [Abstract] [Full Text] [PDF]
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S. Beyth, Z. Borovsky, D. Mevorach, M. Liebergall, Z. Gazit, H. Aslan, E. Galun, and J. Rachmilewitz Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness Blood, March 1, 2005; 105(5): 2214 - 2219. [Abstract] [Full Text] [PDF]
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R. Sarugaser, D. Lickorish, D. Baksh, M. M. Hosseini, and J. E. Davies Human Umbilical Cord Perivascular (HUCPV) Cells: A Source of Mesenchymal Progenitors Stem Cells, February 1, 2005; 23(2): 220 - 229. [Abstract] [Full Text] [PDF]
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B. Bussolati, S. Bruno, C. Grange, S. Buttiglieri, M. C. Deregibus, D. Cantino, and G. Camussi Isolation of Renal Progenitor Cells from Adult Human Kidney Am. J. Pathol., February 1, 2005; 166(2): 545 - 555. [Abstract] [Full Text] [PDF]
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S. Davani, F. Deschaseaux, D. Chalmers, P. Tiberghien, and J.-P. Kantelip Can stem cells mend a broken heart? Cardiovasc Res, February 1, 2005; 65(2): 305 - 316. [Abstract] [Full Text] [PDF]
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R. A. Panepucci, J. L.C. Siufi, W. A. Silva Jr., R. Proto-Siquiera, L. Neder, M. Orellana, V. Rocha, D. T. Covas, and M. A. Zago Comparison of Gene Expression of Umbilical Cord Vein and Bone Marrow-Derived Mesenchymal Stem Cells Stem Cells, December 1, 2004; 22(7): 1263 - 1278. [Abstract] [Full Text] [PDF]
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T. Asahara and A. Kawamoto Endothelial progenitor cells for postnatal vasculogenesis Am J Physiol Cell Physiol, September 1, 2004; 287(3): C572 - C579. [Abstract] [Full Text] [PDF]
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Y. Fukuchi, H. Nakajima, D. Sugiyama, I. Hirose, T. Kitamura, and K. Tsuji Human Placenta-Derived Cells Have Mesenchymal Stem/Progenitor Cell Potential Stem Cells, September 1, 2004; 22(5): 649 - 658. [Abstract] [Full Text] [PDF]
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M. A. Baxter, R. F. Wynn, S. N. Jowitt, J. E. Wraith, L. J. Fairbairn, and I. Bellantuono Study of Telomere Length Reveals Rapid Aging of Human Marrow Stromal Cells following In Vitro Expansion Stem Cells, September 1, 2004; 22(5): 675 - 682. [Abstract] [Full Text] [PDF]
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A. Hermann, R. Gastl, S. Liebau, M. O. Popa, J. Fiedler, B. O. Boehm, M. Maisel, H. Lerche, J. Schwarz, R. Brenner, et al. Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells J. Cell Sci., September 1, 2004; 117(19): 4411 - 4422. [Abstract] [Full Text] [PDF]
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 T. Kinnaird, E. Stabile, M. S. Burnett, and S. E. Epstein Bone Marrow-Derived Cells for Enhancing Collateral Development: Mechanisms, Animal Data, and Initial Clinical Experiences Circ. Res., August 20, 2004; 95(4): 354 - 363. [Abstract] [Full Text] [PDF]
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J. Liu, Q. Hu, Z. Wang, C. Xu, X. Wang, G. Gong, A. Mansoor, J. Lee, M. Hou, L. Zeng, et al. Autologous stem cell transplantation for myocardial repair Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H501 - H511. [Abstract] [Full Text] [PDF]
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G. Kogler, S. Sensken, J. A. Airey, T. Trapp, M. Muschen, N. Feldhahn, S. Liedtke, R. V. Sorg, J. Fischer, C. Rosenbaum, et al. A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential J. Exp. Med., July 19, 2004; 200(2): 123 - 135. [Abstract] [Full Text] [PDF]
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M. F. Pittenger and B. J. Martin Mesenchymal Stem Cells and Their Potential as Cardiac Therapeutics Circ. Res., July 9, 2004; 95(1): 9 - 20. [Abstract] [Full Text] [PDF]
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E. J. Gang, J. A. Jeong, S. H. Hong, S. H. Hwang, S. W. Kim, I. H. Yang, C. Ahn, H. Han, and H. Kim Skeletal Myogenic Differentiation of Mesenchymal Stem Cells Isolated from Human Umbilical Cord Blood Stem Cells, July 1, 2004; 22(4): 617 - 624. [Abstract] [Full Text] [PDF]
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G. D'Ippolito, S. Diabira, G. A. Howard, P. Menei, B. A. Roos, and P. C. Schiller Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential J. Cell Sci., June 15, 2004; 117(14): 2971 - 2981. [Abstract] [Full Text] [PDF]
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J. C. Davila, G. G. Cezar, M. Thiede, S. Strom, T. Miki, and J. Trosko Use and Application of Stem Cells in Toxicology Toxicol. Sci., June 1, 2004; 79(2): 214 - 223. [Abstract] [Full Text] [PDF]
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J. Oswald, S. Boxberger, B. Jorgensen, S. Feldmann, G. Ehninger, M. Bornhauser, and C. Werner Mesenchymal Stem Cells Can Be Differentiated Into Endothelial Cells In Vitro Stem Cells, May 1, 2004; 22(3): 377 - 384. [Abstract] [Full Text] [PDF]
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M. Murga, L. Yao, and G. Tosato Derivation of Endothelial Cells from CD34- Umbilical Cord Blood Stem Cells, May 1, 2004; 22(3): 385 - 395. [Abstract] [Full Text] [PDF]
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S. Neuss, E. Becher, M. Woltje, L. Tietze, and W. Jahnen-Dechent Functional Expression of HGF and HGF Receptor/c-met in Adult Human Mesenchymal Stem Cells Suggests a Role in Cell Mobilization, Tissue Repair, and Wound Healing Stem Cells, May 1, 2004; 22(3): 405 - 414. [Abstract] [Full Text] [PDF]
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M. Bensidhoum, A. Chapel, S. Francois, C. Demarquay, C. Mazurier, L. Fouillard, S. Bouchet, J. M. Bertho, P. Gourmelon, J. Aigueperse, et al. Homing of in vitro expanded Stro-1- or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment Blood, May 1, 2004; 103(9): 3313 - 3319. [Abstract] [Full Text] [PDF]
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M. del Carmen Rodriguez, A. Bernad, and M. Aracil Interleukin-6 deficiency affects bone marrow stromal precursors, resulting in defective hematopoietic support Blood, May 1, 2004; 103(9): 3349 - 3354. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
