Unique multipotent cells in adult human mesenchymal
Yasumasa Kurodaa,1, Masaaki Kitadaa,1, Shohei Wakaoa, Kouki Nishikawab, Yukihiro Tanimuraa, Hideki Makinoshimaa,
Makoto Godac, Hideo Akashia, Ayumu Inutsukab, Akira Niwad, Taeko Shigemotoa, Yoko Nabeshimae,
Tatsutoshi Nakahatad, Yo-ichi Nabeshimae, Yoshinori Fujiyoshib, and Mari Dezawaa,2
aDepartment of Stem Cell Biology and Histology, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan;bDepartment of Biophysics,
Graduate School of Science, Kyoto University, Kyoto 606-8502, Kyoto, Japan;dCenter for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507,
Japan;eDepartment of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; andcJapan Biological
Informatics Consortium (Kyoto Branch Office), Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Edited* by Yoshito Kaziro, Kyoto University, School of Medicine, Kyoto, Japan, and approved March 29, 2010 (received for review October 8, 2009)
We found adult human stem cells that can generate, from a single
cell, cells with the characteristics of the three germ layers. The cells
or bone marrow stromal cells, or directly from bone marrow
in suspension culture that express a set of genes associated with
pluripotency; and can differentiate into endodermal, ectodermal,
and mesodermal cells both in vitro and in vivo. When transplanted
into immunodeficient mice by local or i.v. injection, the cells
integrated into damaged skin, muscle, or liver and differentiated
into cytokeratin 14-, dystrophin-, or albumin-positive cells in the
respective tissues. Furthermore, they can be efficiently isolated as
SSEA-3(+) cells. Unlike authentic ES cells, their proliferation activity
mouse testes. Thus, nontumorigenic stem cells with the ability to
generate the multiple cell types of the three germ layers can be
obtained through easily accessible adult human mesenchymal cells
without introducing exogenous genes. These unique cells will be
beneficial for cell-based therapy and biomedical research.
bone marrow|differentiation|fibroblasts|mesenchymal stem cell|
functional maintenance of organs and to cell renewal, tissue
remodeling, and repair (1, 2). These stem cells are expected to
contribute to regenerative medicine, but this will require elucida-
tion of their stem cell properties to control their proliferation and
differentiation. Among the many kinds of tissue stem cells, hema-
topoietic stem cells and neural stem cells have been characterized
most extensively (i.e., their ability to self-renew and differentiate
into tissue-specific cell types has been clearly demonstrated at the
single-cell level) (3, 4). In contrast, some of the properties of
mesenchymal stem cells remain obscure. For example, one mes-
enchymal cell type, the bone marrow stromal cell (MSC), differ-
entiates into cells of the same mesenchymal lineage, such as
osteocytes, cartilage, and adipocytes, but also differentiates into
cells of other lineages, such as neuronal cells and liver cells, sug-
are thus qualified as multipotent cells (5–8). In most cases, how-
ever, the differentiation was demonstrated in a heterogeneous
population comprising MSCs and not at the single-cell level.
Therefore, it remains under debate whether different subsets of
cells are responsible for differentiation into cell types of different
lineages, such as osteocytes and neuronal cells, or whether a dis-
tinctly multipotent stem cell type exists that is responsible for dif-
ferentiation across all the oligo-lineage boundaries. Furthermore,
their differentiation into cells of all three germ layers has not been
demonstrated at the single-cell level (9–11).
ecent advances in stem cell research have revealed the exis-
In the present study, we demonstrate, at the single-cell level,
that adult human skin fibroblasts, MSCs, and native bone marrow
aspirates contain a distinct type of stem cell that is capable of
cells are indistinguishable from other major mesenchymal cells in
adherent culture, but when they are transferred to suspension
culture, they form characteristic cell clusters that are positive for
Furthermore, they can be efficiently isolated as cells positive for
both SSEA-3, a human pluripotency marker, and CD105, a mes-
enchymal cell marker. The cells exhibit multipotency, but their
proliferation activity is not very high. Furthermore, although
retaining their differentiation ability in vivo, these cells, unlike
authentic ES cells, do not form teratomas in testes of immuno-
deficient mice. Our findings thus suggest that adult human mes-
enchymal cell populations, such as skin fibroblasts and MSCs,
these cells will promote a better understanding of mesenchymal
stem cell properties. Collection and enrichment of these cells
should contribute to improved differentiation efficiency in mes-
enchymal cell populations. Finally, because these cells are easily
accessible, they will be a realistic source of adult human multi-
potent stem cells that are capable of differentiation into cells with
characteristics of all three germ layers without the need to in-
troduce exogenous genes. These cells thus hold great promise for
cell-based therapy and biomedical research.
Analysis of Cell Clusters Generated from Human Mesenchymal Cells.
We found that naive human MSCs (H-MSCs) grown in adherent
culture spontaneously formed characteristic cell clusters at a very
low frequency that appeared similar to clusters formed by human
ES cells at an early stage (Fig. 1A) (12), suggesting that naive H-
MSCs might contain multipotent cells. At a certain size, these
cell clusters stopped growing and had a heterogeneous appear-
to stress, burdens, or damage (13–16). We therefore explored the
possibility of whether stress conditions could be exploited for
a method to enrich the putative stem cells in adult human mes-
enchymal cell populations. We subjected two strains of human
skin fibroblasts (H-fibroblasts) and four strains of H-MSCs to six
Author contributions: M.D. designed research; Y.K., M.K., S.W., K.N., Y.T., H.M., M.G., H.A.,
A.I., A.N., T.S., Y.N., T.N., Y.F., and M.D. performed research; Y.K., M.K., S.W., T.N., Y.-i.N.,
Y.F., and M.D. analyzed data; and M.K., Y.F., and M.D. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1Y.K. and M.K. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 11, 2010
| vol. 107
| no. 19
different stress conditions, including long-term trypsin incubation
(LTT) for 8 or 16 hr (Table S1 and SI Materials and Methods).
Stem cells are often grown in suspension culture, which is an ef-
(17, 18). H-fibroblasts or H-MSCs that survived the stress treat-
ments were therefore suspended in methylcellulose (MC) medium
and Methods) and grown for 7 days (Fig. 1C). Each condition gave
rise to cell clusters with sizes of up to 50–150 μm in diameter (Fig.
1D). Using differentfilters, weseparated thecell clusters according
of the clusters with a diameter larger than 25 μm contained cells
positive for the pluripotency markers Nanog, Oct3/4, SSEA-3,
staining (Fig. S1). We therefore only counted cell clusters larger
potent in the formation of cell clusters in H-fibroblasts, and 8-hr
LTT was most potent in the formation of cell clusters in H-MSCs
(Table S1 and SI Results). As expected, the formed clusters con-
and ALP staining (Fig. 1 K–M). We called these cells multilineage
differentiating stress enduring (Muse) cells because they express
pluripotency markers; as described below, they differentiate into
ectodermal, endodermal, and mesodermal cells; and they endure
through stress conditions. We refer to H-fibroblasts and H-MSCs
treated with 16-hr and 8-hr LTT, respectively, as “Muse-enriched
cell populations” (MEC populations).
To calculate the frequency of Muse-cell-derived cell cluster
(M-cluster; SI Results) formation accurately, MEC populations
derived from both H-fibroblasts and H-MSCs were subjected to
single-cell suspension culture after limiting dilution (Fig. S2 and
SI Materials and Methods), showing that 11.6 ± 1.6% of the cells
in the H-fibroblast–MEC population and 8.1 ± 0.2% of the cells
in the H-MSC–MEC population proceeded to form M-clusters
after 7 days. Naive populations (without LTT) were also exam-
ined and showed that 1.3 ± 0.1% (H-fibroblasts) and 1.1 ± 0.1%
(H-MSCs) of the cells formed M-clusters in single-cell suspen-
sion culture after limiting dilution.
Self-Renewal and Expansion of Muse Cells. After LTT, Muse cells
began to divide after 1–2 days in MC culture and continued to
divide at a rate of ≈1.3 days per cell division until day 8, forming
cell clusters. Cell proliferation gradually slowed by days 11–12
and ceased around day 14, with cell clusters reaching a maximum
size of 150 μm (Fig. S3 and SI Materials and Methods).
When M-clusters formed in single-cell suspension culture after
trypsin treatment and returned to single-cellsuspension culture, the
cells survived but divided very slowly (5–7 days per cell division) or
sometimes not at all (Fig. 1N1). However, transfer of single M-
clusters to adherent culture reinitiated cell proliferation and pro-
duced expanded cells. When cultures that had expanded to ≈3,000–
5,000 cells were dissociated and subjected to single-cell suspension
culture without LTT, 48.0 ± 5.8% (H-fibroblasts) and 40.3 ± 9.1%
(H-MSCs) of the cells formed M-clusters (Fig.1N2).When cultures
LTT to produce MEC populations (Fig. 1N3), 12.3 ± 1.3% (H-
fibroblasts) and 8.5 ± 0.5% (H-MSCs) of these cells formed second
generation M-clusters. We repeated this culture cycle, consisting of
cell generation showed similar behavior and a similar frequency of
clusters were still positive for pluripotency markers and ALP stain-
ing. Furthermore, karyotypes of cells expanded from M-clusters did
not show detectable abnormalities (Fig. S4 and SI Results). In con-
clusion, the proliferation activity of Muse cells is not very high, and
a defined size. Nevertheless, the proliferation of Muse cells can be
reinitiated by transfer to adherent culture, which is followed by the
of Muse cells for self-renewal and proliferation.
Differentiation of M-Clusters. Toanalyzetheirdifferentiationability,
single M-clusters formed in single-cell suspension culture after
limiting dilution were transferred onto gelatin-coated dishes. After
7 days of culture, immunocytochemistry revealed cells positive for
neurofilament-M [an ectodermal marker; the ratio of positive
cells was 3.5 ± 0.5% (H-fibroblasts) and 3.7 ± 0.6% (H-MSCs)],
α-smooth muscle actin [α-SMA; mesodermal, 12.2 ± 1.8% (H-
[endodermal, 5.5 ± 0.1% (H-fibroblasts) and 3.4 ± 0.6% (H-
MSCs)], or desmin [mesodermal, 14.2 ± 0.4% (H-fibroblasts) and
first- and third-generation M-clusters confirmed that these cells
that occur spontaneously in adherent cultures of naive H-MSCs. (C and D) MC
culture of H-fibroblasts on day 7 showing an M-cluster (C, arrow). Immu-
nocytochemical localization of Nanog (E and F), Oct3/4 (G), Sox2 (H), PAR4
(I), and SSEA-3 (J) in M-clusters formed by H-fibroblasts (E, I, and J) and H-
MSCs (F, G, and H). ALP(+) human ES cells (K), M-cluster (H-fibroblast) (L), and
naive H-fibroblasts (M). (N) Schematic diagram of the self-renewal of Muse
cells. (Scale bars: A–C, 100 μm; D–M, 50 μm.)
Characterization of M-clusters. (A and B) Characteristic cell clusters
| www.pnas.org/cgi/doi/10.1073/pnas.0911647107 Kuroda et al.
expressed α-fetoprotein (endodermal) and GATA6 (endodermal),
(mesodermal), whereas these markers were not clearly detected
in naive fibroblasts and MSC populations (Fig. 2F).
We injected MEC populations or M-clusters into the testes of
immunodeficient mice to test whether they form teratomas. None
of the testes injected with MEC populations or M-clusters formed
teratomas for up to 6 months, and most of the testes were not
significantly larger than control testes (Fig. 2G and SI Results). In
the MEC- or M-cluster-injected testes, cells positive for human
mitochondria and for ectodermal (neurofilament), endodermal
(α-fetoprotein), and mesodermal (SMA) lineage markers were
detected (Fig. 2 H–M and Fig. S5).
injection of GFP-labeled H-MSC–MEC population), gastrocne-
mius muscle (i.v. injection of GFP-H-fibroblast–MEC population),
or liver (i.v. injection of GFP-H-fibroblast–MEC population) of
immunodeficient mice. In regenerating skin, after 2 weeks, 79.5 ±
2.0% of the transplanted cells in the epidermis also expressed
cytokeratin 14 (Fig. 3A). GFP(+) cells were also incorporated into
regenerating muscle. After 2 weeks, the nuclei of these cells were
expressed human dystrophin (Fig. 3 B and C). Some of the trans-
planted cells expressed satellite cell marker Pax7 (Fig. 3B). In the
antitrypsin (87.6 ± 3.0%; Fig. 3 D and E). These data suggest that
MEC populations can differentiate into ectodermal, endodermal,
and mesodermal lineage cells in vivo.
M-Cluster Formation Directly from Native Bone Marrow Aspirate. The
experiments described so far were performed with Muse cells and
M-clusters derived from cell cultures, which may have acquired
characteristics that differ from those of cells in situ. Mesenchymal
marrow, which can be collected directly from native tissue without
culturing.Wetherefore testedwhether cells directlycollected from
humanbone marrow(hBM)wouldalsobe abletoformM-clusters.
Isolated mononucleated cells were either subjected directly to MC
culture (naive hBM-MC) or to 8-hr LTT before MC culture (8-hr
hBM-MC). After 7 days, 8-hr hBM-MC formed M-clusters at
a frequency of 0.3 ± 0.04%, ≈60–75 times higher than that of naive
hBM-MC (0.004 ± 0.001%) (Fig. 3F). M-clusters from both naive
of cells expanded from single M-clusters on gelatin-coated dishes
showed expression of α-fetoprotein, GATA6, MAP-2, and Nkx2.5
(Fig. 3H). These results suggest that M-clusters can be formed di-
rectly from hBM and that they can be enriched by 8-hr LTT.
Because culturing can change the composition of cell populations,
chemistry of neurofilament-M (NF) (A), α-SMA (B), α-fetoprotein (α-FP) (C),
cytokeratin 7 (CK7) (D), and desmin (E) in cells derived from a single M-
cluster (H-fibroblasts). (F) RT-PCR analysis of naive cells and first- and third-
generation M-clusters (first and third clusters) derived from H-fibroblasts.
Positive controls were human fetus liver (Liver) for α-FP and whole human
embryo (Embryo) for GATA6, MAP-2, and Nkx2.5. (G–M) Testes of immu-
nodeficient mice injected with cells. (G) Uninjected testes (intact) and testes
injected with mouse ES cells (8 weeks), mouse embryonic fibroblast (MEF)
cells (8 weeks), and M-clusters (6 months). Immunohistochemistry of NF (H),
α-FP (I), and SMA (J) in testes injected with MEC populations and M-clusters.
(K) Double-labeling of human mitochondria (green) and SMA (red). The
tube-like structure (L) was positive for human mitochondria in the adjacent
section (M; red). (Scale bars: A–E and H–L, 50 μm; M, 20 μm.)
Differentiation of Muse cells in vitro and in testes. Immunocyto-
the regenerating epidermis (2 weeks). (B) Two weeks after i.v. injection, GFP(+)
cells with central nuclei were seen in cardiotoxin-injected cutaneous muscle.
Transplanted GFP(+) cells (arrow) and host cells [GFP(−), arrowhead] that
human dystrophin (h-Dystrophin; red). Four weeks after i.v. injection, most of
(D, red) or human antitrypsin (E, red). (F–H) Formation of M-clusters from bone
marrow-derived mononucleated cells. (F) M-clusters formed with 8-hr LTT (8-hr
hBM-MC, day 7). (G) ALP(+) cells in 8-hr hBM-MC (day 7). (H) RT-PCR of naive H-
MSCs (Naive 1 and Naive 2); M-clusters formed with 8-hr LTT (8-hr hBM) or
without LTT [Naive hBM (N-hBM)]. Positive controls were human fetus liver
(Liver) for α-fetoprotein (α-FP) and whole human embryo (Embryo) for GATA6,
MAP-2, and Nkx2.5. (Scale bars: A, B, E, F, and G, 50 μm; C and D, 100 μm.)
Transplantation of Muse cells and M-cluster formation from bone
Kuroda et al.PNAS
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cells in stable culture may have a different propensity to form M-
hBM aspirate in adherent culture to collect primary MSCs and
subjected the cells directly to MC culture without 8-hr LTT. This
of 0.3 ± 0.08%. When primary MSCs were further cultured to the
second and fifth passages, the frequency of M-cluster formation
without LTT increased up to 0.5 ± 0.04% and 0.9 ± 0.1%, re-
spectively. Consistent with this finding, 1.3 ± 0.1% of naive H-
fibroblasts and 1.1 ± 0.1% of naive H-MSCs formed M-clusters in
single-cell suspension culture as described above. These results
vitro culture and the subculture procedures.
Bone marrow contains many cell types, including MSCs, he-
cells from hBM aspirate and subjected them to magnetic affinity
cell sorting (MACS) using antibodies against CD34 and CD117
[markers for hematopoietic cells (20)] and CD105 [marker for
fraction produced few M-clusters, but the CD34−/117−/105+
105−fraction (SI Results). This result suggests that the majority of
Characteristic Features of Muse Cells. FACS analysis revealed that
among tested surface markers, MEC populations showed a sub-
stantially increased number of cells that were SSEA-3(+) [a human
and SI Results). The percentage of SSEA-3(+) cells detected by
4A] and immunocytochemistry [0.7 ±0.1% (H-fibroblasts); Fig. 4B]
cluster formation in single-cell suspension culture (≈1%), as de-
cellsdetectedby FACS[11.6±0.15% (H-MSCs) and8.6±0.0.32%
cluster formation in single-cell suspension culture [11.6 ± 1.6% (H-
fibroblasts) and 8.1 ± 0.2% (H-MSCs)] as stated. Furthermore, the
in cultured H-fibroblasts using immunocytochemistry (Fig. 4 D and
E). We therefore used FACS to separate SSEA-3(+) and (−) cells
from MEC populations of both H-fibroblasts and H-MSCs and
dilution. The result showed that 56.5 ± 3.2% (H-MSCs) and 60.0 ±
4.5% (H-fibroblasts) of the SSEA-3(+) cells generated M-clusters,
The importance of SSEA-3(+) cells was also seen in the trans-
plantation experiments. In contrast to the integration and differ-
entiation of MEC populations in damaged skin, muscle, and liver
(Fig.3 A–E),transplantationofSSEA-3(−) populations resultedin
were positive for the tissue markers (Fig. S6 and SI Results).
Of note, at the scale of 3,000–5,000 cells, only ≈45.0 ± 3.2%
(H-fibroblasts) of the cells that expanded from a single FACS-
sorted SSEA-3(+) cell were SSEA-3(+) (Fig. 4C). This result
suggests that proliferation of Muse cells may give rise to Muse
cells and non-Muse cells.
The possibility remains that Muse cells are artificially induced by
LTT. As described above, the majority of Muse cells exist in the
bone marrow’s CD105(+) cell fraction. Furthermore, SSEA-3(+)
Muse cells directly from adult hBM aspirates by isolating them as
SSEA-3/CD105 double-positive cells. Double-positive cells, which
constituted 0.04 ± 0.008% of bone marrow-derived mononucleated
cells, were directly subjected to RT-PCR, which showed the expres-
sion of Nanog, Oct3/4, and Sox2 in these cells (Fig. 4F). Isolated
SSEA-3+/CD105+cells were further subjected to single-cell sus-
1.2% of the cells (corresponding to 0.003–0.005% of the mono-
nucleated cells) formed M-clusters that were ALP(+). Single M-
clusters were then again expanded in adherent culture to 3,000 cells
cells, 33.5 ± 3.1% formed second-generation M-clusters, and RT-
PCR of the cells that expanded from a single M-cluster on gelatin-
coated dishes indicated that the cells expressed α-fetoprotein,
GATA6, MAP-2, and Nkx2.5 (Fig. 4G), suggesting that cells with
properties consistent with those of Muse cells reside in adult hBM.
We isolated a specific type of human mesenchymal stem cell (i.e.,
of all three germ layers from a single cell. Muse cells are (i) stress
tolerant; (ii) indistinguishable from general mesenchymal cells in
that are positive for pluripotency markers and ALP staining; (iv)
tissues as cells positive for both CD105 and SSEA-3.
To investigate whether Muse cells exist in native tissues or
whether LTT induces cells to acquire properties of multipotent
stem cells, we isolated SSEA-3/CD105 double-positive cells di-
rectly from hBM aspirates and subjected them to single-cell sus-
hBM. LTT is thus not necessary for collecting Muse cells but is
a method to enrich them.
The properties of several kinds of stem cells present in mes-
enchymal tissue, such as neural crest-derived stem cells (NCSCs)
that exist both in the bone marrow and skin, MSCs, skin-derived
precursors (SKPs), perivascular cells, and adipose-derived stem
in naive cells (Naive) and MEC populations (Muse) derived from H-fibroblasts
and H-MSCs. SSEA-3(+) cells (red) in a naive population (B) and in cells ex-
panded from a single M-cluster derived from a FACS-sorted SSEA-3(+) cell (C),
both from H-fibroblasts. Immunocytochemistry of Oct3/4 (D, green, Sox2 (E,
green), and SSEA-3 (D and E, red) in Muse cells derived from H-fibroblasts. (F)
RT-PCR of Oct3/4, Sox2, and Nanog in directly isolated SSEA-3+/CD105+cells
from bone marrow, human ES cells for a positive control, and the template
second-generation M-clusters (2nd M-cluster) from bone marrow-derived
mononucleated cells. Positive controls were human fetus liver (Liver) for
α-fetoprotein (α-FP) and whole human embryo (Embryo) for GATA6, MAP-2,
and Nkx2.5. (Scale bars: B and C, 100 μm; D, 10 μm; E–G, 5 μm.)
| www.pnas.org/cgi/doi/10.1073/pnas.0911647107Kuroda et al.
cells, were recently analyzed and described (11, 21–24). Of note, Download full-text
SKPs can be clonally expanded and serve as dermal stem cells for
use in skin homeostasis and repair (11). NCSCs can also be
clonally expanded, and they may contribute to nerve repair (22).
Although, these stem cells can differentiate into ectodermal
(e.g., neural marker-positive cells) and mesodermal (e.g., SMA-
positive cells, osteocytes, chondrocytes) lineage cells (11, 21–24),
their differentiation into representatives of all three germ layers
has not been reported. Muse cells are unique among mesen-
chymal stem cells in that they are able to differentiate not only
into ectodermal and mesodermal cells but into endodermal cells.
That is, a single Muse cell can generate cells representative of
each of the three germ layers. Considering that multilineage
differentiation of MSCs, such as into neuronal cells, muscle cells,
and liver cells, has been reported (5–8), it is possible that Muse
cells contribute to such multilineage differentiation of MSCs.
Muse cells may have practical advantages for regenerative medi-
cine, such as their easy accessibility and differentiation potential.
They are not tumorigenic but retain differentiation ability in vivo
after transplantation. In fact, they expressed endodermal, ectoder-
mal, and mesodermal lineage markers after injection into mouse
testes; integrated into damaged skin, muscle, and liver tissue; and
differentiated into cells expressing the respective tissue markers
(endodermal). Moreover, the multipotency of Muse cells does not
can be isolated from skin and bone marrow, which are accessible
a very small number of stem cells, and like other stem cells, the
proportion of Muse cells in bone marrow-derived mononucleated
cells is very small. However, large numbers of Muse cells can be
obtained from mesenchymal cell populations by simply cycling the
cells through a series ofculturing steps, namely, Muse-cell selection
followed by formation of M-clusters in suspension culture and ex-
pansion of the cells in adherent culture.
Muse cells did not show characteristics of tumorigenic pro-
liferation, and, consistently, they did not develop into teratomas
in mice testes. It has recently been reported that epiblast stem
cells cultured under certain conditions also did not form ter-
atomas in testes, even though they showed pluripotency in vitro
(25). This finding suggests that even pluripotent cells do not al-
ways show the formation of teratomas in testes. Furthermore, if
Muse cells are normally maintained in adult human tissues, such
as in skin fibroblasts, their proliferation must be strictly regu-
lated; otherwise, they would easily form tumors in virtually every
part of the body.
of Muse cells in fibroblasts and MSCs will require further study.
Materials and Methods
Culture Cells. Two strains of human skin fibroblasts and four strains of H-MSCs
were maintained at 37 °C in α-minimum essential medium (α-MEM) con-
taining 10% (vol/vol) FBS and 0.1 mg/mL kanamycin. Mononucleated cells
were collected from six hBM aspirates using Lymphoprep Tubes (Axis-Shield
PoC AS). For selection of FBS, an H-MSC clone was plated onto a 24-well dish
at a density of 1.5 × 104cells per cm2. Serum lots (ES cell grade; HyClone)
were checked by adding a sample from each lot to a well at a concentration
of 10% (vol/vol) in α-MEM and cultured for 1–2 weeks. The serum in the well
that showed the highest frequency of spontaneous cell cluster formation, as
shown in Fig. 1 A and B, was chosen for further experiments.
Generation of MEC Populations and M-Clusters. MEC populations were pro-
duced by treating cells with LTT (16 hr for H-fibroblasts and 8 hr for H-MSCs),
followed by vortexing at 1,800–2,200 rpm for 3 min (MS1 Minishaker, IKA
Works, Staufen, Germany) and centrifugation at 740 × g for 15 min. To
produce M-clusters, individual cells were cultured in MC or in single-cell
suspension culture. For MC culture, culture dishes were first coated with
polyHEMA (P3932; Sigma) to avoid attachment of cells to the bottom of the
dish. MC (MethoCult H4100; StemCell Technologies) was diluted in 20% (vol/
vol) FBS in α-MEM to a final concentration of 0.9%. The cell concentration in
the semisolid MC medium was adjusted to be 8 × 103cells per milliliter. Cells
and MC were mixed thoroughly by gentle pipetting, and the mixture was
transferred to a polyHEMA-coated dish. At this concentration, the cell-to-cell
distance was sufficiently large to minimize cell aggregation. For single-cell
suspension culture, MEC populations were subjected to a limiting dilution
with 10% (vol/vol) FBS in α-MEM and single cells were plated into each well
coated with polyHEMA. The frequency of M-cluster formation was calcu-
lated from three experiments for each strain, with a minimum of 250 wells
Detailed protocols for cell culture, stress conditions, ALP staining, im-
munocytochemistry, immunohistochemistry, transplantation experiments,
RT-PCR, karyotyping, MACS sorting, and FACS analysis are provided in
ACKNOWLEDGMENTS. We thank Dr. Thomas Walz (Harvard Medical School)
for proofreading the manuscript and Dr. Hiroshi Hamada (Osaka University,
Japan) for providing antibodies. We thank the late Keiji Takita, Director
General of the Japan New Energy and Industrial Technology Development
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Kuroda et al.PNAS
| May 11, 2010
| vol. 107
| no. 19