Pluripotency of mesenchymal stem cells derived from adult marrow

Nature AOP, published online 20 June 2002; doi:10.1038/nature00870. Texto completo. Se requiere registro:http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/vaop/ncurrent/full/nature00870_fs.html

* Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota
55455, USA
‡ Division of Hematology, Oncology and Transplantation, Department of Medicine,
University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA
§ Department of Microbiology, Center for Immunology, University of Minnesota Medical
School, Minneapolis, Minnesota 55455, USA
  Department of Neurosurgery, University of Minnesota Medical School, Minneapolis,
Minnesota 55455, USA
¶ Department of Genetics, Cell Biology and Development, University of Minnesota Medical
School, Minneapolis, Minnesota 55455, USA
† These authors contributed equally to this work

Correspondence and requests for materials should be addressed to C.M.V. (e-mail:
[email protected]).
 

We report here that cells co-purifying with mesenchymal
stem cells—termed here multipotent adult progenitor cells or
MAPCs—differentiate, at the single cell level, not only into
mesenchymal cells, but also cells with visceral mesoderm,
neuroectoderm and endoderm characteristics in vitro. When
injected into an early blastocyst, single MAPCs contribute to
most, if not all, somatic cell types. On transplantation into a
non-irradiated host, MAPCs engraft and differentiate to the
haematopoietic lineage, in addition to the epithelium of liver,
lung and gut. Engraftment in the haematopoietic system as
well as the gastrointestinal tract is increased when MAPCs
are transplanted in a minimally irradiated host. As MAPCs
proliferate extensively without obvious senescence or loss of
differentiation potential, they may be an ideal cell source for
therapy of inherited or degenerative diseases.

Embryonic stem (ES) cells are pluripotent cells derived from the
inner cell mass of the blastocyst that can be propagated
indefinitely in an undifferentiated state. ES cells differentiate to
all cell lineages in vivo and differentiate into many cell types in
vitro. Although ES cells have been isolated from humans1, their
use in research as well as therapeutics is encumbered by ethical
considerations2. Stem cells also exist for most tissues, including
haematopoietic3, neural4, gastrointestinal5, epidermal6, hepatic7
and mesenchymal stem cells8. Compared with ES cells,
tissue-specific stem cells have less self-renewal ability and,
although they differentiate into multiple lineages, they are not
pluripotent.

Until recently, it was thought that tissue-specific stem cells
could only differentiate into cells of the tissue of origin;
however, recent studies suggested that tissue-specific stem cells
can differentiate into lineages other than the tissue of origin.
After transplantation of bone marrow or enriched
haematopoietic stem cells (HSC), skeletal myoblasts9, 10,
cardiac myoblasts11, 12, endothelium11-14, hepatic and biliary
duct epithelium15-17, lung, gut and skin epithelia18, and
neuroectodermal cells of donor origin have been detected19-22.
Some but not other studies demonstrated that neural stem
cells23, 24 as well as muscle cells25, 26 may differentiate into
haematopoietic cells. When injected into a blastocyst, neural
stem cells contribute to a number of tissues of the chimaeric
mouse embryo27; however, most studies did not conclusively
demonstrate that a single tissue-specific stem cell differentiates
into functional cells of multiple tissues.

We identified a rare cell within human bone marrow
mesenchymal stem cell cultures that can be expanded for more
than 80 population doublings. This cell differentiates not only
into mesenchymal lineage cells but also endothelium28, 29 and
endoderm30. We show that cells capable of differentiating in
vitro to cells of the three germ layers can be selected from
rodent bone marrow. These cells contribute to most somatic
tissues when injected into an early blastocyst and engraft in vivo,
where they differentiate into tissue-specific cell types in
response to cues provided by different organs.

Culture of undifferentiated mouse and rat MAPCs
To isolate MAPCs from murine bone marrow, we used methods
identical to those used for human (h)MAPCs28, except that
murine (m)MAPCs, but not hMAPCs, required leukaemia
inhibitory factor (LIF) for expansion. (A detailed description of
the culture method can be found in Supplementary Information
Table 1.) We have been able to culture several mMAPC
populations for more than 120 population doublings (Fig. 1a).
The phenotype of mMAPCs in fresh bone marrow is unknown.
The phenotype of cultured mMAPCs is CD34, CD44, CD45,
c-Kit, and major histocompatibility complex (MHC) class I and
II negative; mMAPCs express low levels of Flk-1, Sca-1 and
Thy-1, and higher levels of CD13 and stage-specific antigen I
(SSEA-I)1 (Fig. 1b). The morphology and phenotype were
similar after 30 to more than 120 population doublings
(Supplementary Information Fig. 1). The mMAPC phenotype is
similar to that of hMAPC28 but different from that of murine
haemopoietic stem cells with 'transdifferentiation' potential11, 17,
18, 25. mMAPCs were 8–10 µm in diameter with a large nucleus
and scant cytoplasm (Supplementary Information Fig. 1a).
Average telomere length (ATL) of mMAPCs cultured for 40
population doublings was 27 kilobases (kb); when re-tested after
102 population doublings, ATL remained unchanged (Fig. 1c).
 

                    Figure 1 Characteristics of mMAPCs.
                      Full legend

                    High resolution image and legend (73k)
 

Similar results were obtained when we isolated and cultured
MAPCs from bone marrow of Sprague–Dawley rats (n = 3). We
have expanded rat (r)MAPCs for more than 100 population
doublings (Supplementary Information Fig. 2a). Successful
culture of rMAPCs required addition of LIF to epidermal growth
factor (EGF) and platelet-derived growth factor (PDGF)-BB.
rMAPCs were MHC class I and class II negative, CD44 negative
(data not shown), expressed high levels of telomerase, and the
telomeres have not shortened in culture for 100 population
doublings (Supplementary Information Fig. 2b).

The finding that rodent but not hMAPCs require addition of LIF
to the culture medium is similar to results for ES cells: human
ES cells seem to be LIF-independent31 whereas murine ES cells
require LIF for ex vivo maintenance32. Quantitative reverse
transcription polymerase chain reaction (Q-RT–PCR) showed
that two transcription factors important in maintaining
undifferentiated ES cells, Oct-4 (ref. 33) and Rex-1 (ref. 34),
were expressed in mMAPCs: Rex-1 at levels similar to mouse
ES cells and Oct-4 at levels 1,000-fold lower than ES cells (Fig.
1d).

In vitro differentiation of single mMAPCs and rMAPCs
Approximately 1% of wells seeded with ten CD45- TER119-
bone marrow monocular cells (BMMNCs) yielded continuous
growing cultures, suggesting that 1 out of 1,000 CD45- TER119-
BMMNCs is capable of initiating MAPC cultures, and that
progeny generated from ten cells is probably derived from one
cell. To definitively prove that a single cell gives rise to
continuous growing cultures and differentiated progeny, we used
retroviral marking (Fig. 2; see also Supplementary Information
Table 2). After sub-cloning at ten cells per well, three murine
and two rat enhanced green fluorescent protein-expressing
(eGFP+) cell populations were selected and culture-expanded for
more than 100 population doublings. One hundred per cent of
cells continued to express GFP after expansion. Southern blot
analyses showed that a single retroviral insert was detected in
one mMAPC and one rMAPC population derived from ten initial
cells (Fig. 2). The same single retroviral insert was also seen in
seven subclones of the rMAPC population, demonstrating that
progeny of only a single eGFP-transduced mMAPC or rMAPC
gave rise to the continuous growing populations35, 36. We
sequenced the genomic flanking region 3' from the retrovirus
using splinkerette PCR, which detects a single retroviral insert in
as few as 5  103 cells37. A single retroviral insertion site was
detected in the mMAPC and rMAPC population (Supplementary
Information Table 2). Presence of the flanking region was
confirmed by PCR using primers designed in the murine stem
cell virus (MSCV) long terminal repeat (LTR) and in the mouse
or rat genomic flanking sequence 3' of the insertion site.
 

                     Figure 2 Single cell origin of mMAPC and
                     rMAPC cultures initiated from ten cells per
                     well, and their differentiated progeny (see
                     also Supplementary Information Table 2).
                       Full legend

                     High resolution image and legend (35k)
 

We next tested the in vitro differentiation ability of mMAPCs
and rMAPCs by adding cytokines chosen on the basis of what
has been described for differentiation of hMAPC or ES cells to
mesoderm, neuroectoderm and endoderm. Differentiation
required that MAPCs be replated at 1–2  104 cells cm-2 in
medium without serum, EGF, PDGF-BB and LIF, but with
lineage-specific cytokines. Studies were done using two
independently derived ROSA26, two C57BL/6 and one rat
MAPC population expanded for 40–120 population doublings, as
well as with eGFP-transduced clonal mMAPCs (repeated after
50, 80 and 120 population doublings) and clonal rMAPCs
(repeated after 50 and 80 population doublings). Differentiation
was similar when MAPCs cultured for 40 to more than 100
population doublings were used. No differences were seen
between transduced and untransduced cells. Most data shown are
therefore from the clonal eGFP+ mMAPCs (Fig. 3) and clonal
eGFP+ rMAPCs (Supplementary Information Fig. 3).
 

                     Figure 3 In vitro differentiation of mMAPCs
                     to endothelium, neuroectoderm and
                     endoderm.   Full legend

                     High resolution image and legend (97k)
 

As an example of mesoderm, we induced differentiation to
endothelium. Undifferentiated mMAPCs or rMAPCs were
negative for CD31, CD62E or von Willebrand factor (vWF)
(data not shown), but expressed low levels of Flk-1 (Fig. 1b).
When mMAPCs (Fig. 3) or rMAPCs (Supplementary
Information Fig. 3) were cultured on fibronectin (FN) with
10 ng ml-1 vascular endothelial growth factor (VEGF)-B for 14
days, more than 90% of MAPCs acquired an endothelial
phenotype and expressed CD31, Flk-1 and vWF. All cells
staining positive for vWF also expressed eGFP, whereas a small
population of eGFP+ 'fibroblast-like' cells not staining positive
for vWF remained in the culture (Fig. 3a–h).

Neuroprogenitors can be expanded with PDGF-BB and induced
to differentiate by removal of PDGF and addition of basic
fibroblast growth factor (bFGF)38. Therefore, we cultured
mMAPCs (Fig. 3) and rMAPCs (Supplementary Information
Fig. 3) in FN-coated wells without PDGF-BB and EGF, but with
100 ng ml-1 bFGF38. After 14 days, 15  4% of MAPCs acquired
morphologic and phenotypic characteristics of astrocytes (glial
acidic fibrillary protein (GFAP) + ), 12  3% of
oligodendrocytes (galactocerebroside (GalC)+) and 68  9% of
neurons (neurofilament-200 (NF-200)+). NF-200, GFAP or GalC
were never found in the same cell. More than 90% of eGFP+
cells were labelled with neuroectodermal markers. Quantitative
RT–PCR of mMAPCs treated with bFGF confirmed expression
of neuroectodermal genes (Supplementary Information Table 3).
After culture with bFGF, levels of Otx2 messenger RNA
increased more than 50-fold by day 2 and became maximal by
day 5. On day 4, Otx1 mRNA was upregulated 3–5-fold, and on
day 5 levels of Pax2, Pax5 and nestin mRNA increased
400–800-fold over undifferentiated mMAPCs39. When
mMAPCs were cultured sequentially with 100 ng ml-1 bFGF,
10 ng ml-1 FGF-8 and finally 10 ng ml-1 brain-derived
neurotrophic factor (BDNF), a more mature phenotype was seen
(Fig. 3i–q). Thirty percent of cells expressed markers of
dopamine-containing neurons (dopa-decarboxylase (DDC) and
tyrosine hydroxylase (TH) positive), 20% of
serotonin-containing (serotonin positive) neurons and 50% of
 -aminobutyric acid (GABA)-containing (GABA positive)
neurons. Neuron-like cells became polarized, as Tau and MAP2
were expressed in axonal and somatodendritic compartments,
respectively (Fig. 3m).

We next tested whether mMAPCs (Fig. 3) or rMAPCs
(Supplementary Information Fig. 3) differentiate to endodermal
cells. As described more extensively elsewhere30, when replated
on matrigel with 10 ng ml-1 FGF-4 and 20 ng ml-1 hepatocyte
growth factor (HGF), approximately 60% of mMAPCs (Fig.
3a–h) or rMAPCs acquired epithelioid morphology and 10% of
cells became binucleated. Sixty per cent of cells stained positive
for albumin, cytokeratin (CK)18, and HNF-1. Figure 3a–h shows
that all cells staining positive for CK18 were also eGFP+. We
recently showed that these epithelioid cells have functional
characteristics of hepatocytes, including urea and albumin
production, phenobarbital-inducible p450, gluconeogenesis and
low-density lipoprotein uptake30.

Southern blot analyses and flanking region PCR demonstrated
that differentiated eGFP+ mMAPCs and rMAPCs contained the
identical single retroviral insert found in undifferentiated
MAPCs (Fig. 2 and Supplementary Information Table 2). As
100% of differentiated cells were eGFP+ (Fig. 3a–h) and only a
single retroviral insertion site was present, these studies show
that a single MAPC differentiates into cells with morphologic,
phenotypic and functional characteristics of cells representing
the three germ layers.

Single MAPCs contribute to most somatic tissues
To further determine the extent of differentiation of MAPCs, we
assessed their ability to contribute to various tissues by
introducing MAPCs into an early blastocyst. One or 10–12
ROSA26 MAPCs obtained after 55–65 population doublings
were microinjected into 3.5-day-old blastocysts of C57BL/6
mice. Blastocysts were transferred to foster mothers, and mice
were allowed to develop and be born (Table 1). The number of
litters born and animals per litter were in line with the birth rate
seen in other studies during this period. Animals born from
microinjected blastocysts were of similar size as normal animals
and did not display overt abnormalities.

Chimaerism was assessed by comparing levels of Neo/-Gal40 in
tail clippings of 4-week-old animals with that of tissue of
ROSA26 mice using Q-PCR. Chimaerism could be detected in
80% of mice derived from blastocysts in which 10–12 mMAPCs
were injected and in 33% of mice derived from blastocysts
microinjected with 1 mMAPC (Table 1). In both sets of animals,
chimaerism ranged between 0.1% and 45%. Absence of
chimaerism in some of the microinjected blastocysts may
indicate that mMAPCs are not completely homogeneous.
Alternatively, technical problems with injection of a single cell
may be responsible for the failure of 66% of single mMAPCs to
contribute to development of the mouse.

Animals were killed at 6–20 weeks. Some mice were frozen in
liquid nitrogen, thin whole-mouse sections cut as described41,
and stained with 5-bromo-4-chloro-3-indolyl--D-galactoside
(X-gal). Shown in Fig. 4i is a representative animal,
non-chimaeric as determined by tail clip analysis, derived from
a blastocyst in which a single MAPC was injected. No X-gal
staining was seen. In contrast, the animal in Fig. 4j, which was
45% chimaeric by tail clip analysis, had the contribution of a
single ROSA26-derived MAPC to many somatic tissues.
 

           Figure 4 Chimaerism detection by X-gal staining and
           anti--gal staining in animals generated from blastocysts
           microinjected with a single ROSA26 MAPC (see also
           Table 1 and Supplementary Information Fig. 4).
           Full legend

           High resolution image and legend (276k)
 

We also collected multiple organs separately and determined the
presence of mMAPC-derived cells by X-gal staining (Fig. 4a–h;
see also Supplementary Information Fig. 4) and staining with an
anti--gal fluorescein isothiocyanate (FITC) antibody (Fig. 5).
Chimaeric animals that had Neo/-Gal+ cells, as determined by
PCR in the tail clip analysis, had a contribution of the
ROSA26-derived MAPC to many tissues, including brain, retina
(not shown), lung, myocardium, skeletal muscle, liver, intestine,
kidney, spleen, bone marrow, blood (not shown), and skin (as
shown by X-gal staining, Fig. 4a–h; see also Supplementary
Information Fig. 4). -gal+ cells expressed markers typical for
the tissue in which they had incorporated. -gal+ cells in bone
marrow, spleen and blood co-stained for CD45, Gr-1, Mac-1,
CD19 and CD3 antigens (Fig. 5). Because of the haematopoietic
chimaerism, we used triple-colour immunofluorescence to
assure that -gal+ cells in solid organs were not mere
haematopoietic cells. -gal+ cells co-stained for pan-CK in the
lung and intestine, and for CK18 in the liver (Fig. 5). We also
detected CD45+ CK- -Gal+ cells in these organs. However, no
cell co-stained for all three antigens (that is, -gal, CD45 and
CK). -gal+ cells co-stained for dystrophin in skeletal muscle
and cardiac troponin-I in the myocardium (Fig. 5). -gal+ cells
gave rise to neurons (Neu-N+) and astrocytes (GFAP+)
throughout the entire brain, including the cortex, striatum,
hippocampus, thalamus and cerebellum (Fig. 5). In the cingulate
cortex, -gal+ neurons and astrocytes were present, whereas in
the underlying corpus callosum, -gal+ astrocytes and presumed
oligodendroglia were found. In the hippocampus, most of the
granule cells of the dentate gyrus and pyramidal cells of the
hilus were -gal+, and were interspersed with -gal+,
MAPC-derived astrocytes.
 

                     Figure 5 Immunofluorescence staining of
                     individual organs from a 45% chimaeric
                     mouse.   Full legend

                     High resolution image and legend (141k)
 

It has been reported that after injection of ROSA26-derived
neural stem cells into murine blastocysts, LacZ-expressing cells
are found with varying degrees not only in the brain, but also in
some mesodermal and endodermal tissues of the chimaeric
mouse embryo27. Our results confirm and extend these studies,
as we show that single MAPCs generate balanced chimaeras,
that is, contribute to most somatic cell types, and that
chimaerism can be detected not only in mouse embryos, but also
in mice that were 6–20 weeks old. Although some LacZ+ cells
were seen in gonads, we have not yet performed breeding
experiments to test whether MAPCs contribute to the germ line.

mMAPCs engraft and differentiate in tissue-specific cells
We next tested whether mMAPCs infused intravenously in
post-natal animals engraft and differentiate in tissue-specific
cells. To avoid rejection by recipient animals, undifferentiated
ROSA26 MAPCs were injected by means of the tail vein into
non-irradiated or irradiated (250 cGy) 6–8-week-old non-obese
diabetic/severe combined immunodeficient (NOD/SCID)
recipients. Engraftment of -gal/Neo-containing cells was tested
by immunohistochemistry (for -gal) and Q-PCR (for Neo),
4–24 weeks after transplantation (Table 2 and Fig. 6).
Engraftment, defined as more than 1% -gal+ cells by
immunofluorescence and/or Q-PCR, was seen in haematopoietic
tissues (blood, bone marrow and spleen) and epithelium of lung,
liver and intestine of all recipient animals, and was similar in
animals analysed 4–24 weeks after transplantation.
 

                     Figure 6 Engraftment and in vivo
                     differentiation of mMAPCs.   Full legend

                     High resolution image and legend (134k)
 

 -gal+ cells in bone marrow and spleen co-stained for CD45
(Fig. 6). Thirty-eight to 62% of bone marrow -gal+ cells
co-stained for Gr-1, 9–27% for Mac-1, 18–31% for CD19, and
4–11% for TER119 (Supplementary Information Table 4).
Similar results were seen for blood (not shown). No -gal+ CD3+
T cells were seen in blood, bone marrow or spleen even though
 -gal+ CD3+ T cells were present in chimaeric mice. The reason
for this is unknown. Engraftment in the spleen occurred mostly
as clusters of donor cells, consistent with the hypothesis that
when MAPCs home to the spleen, they proliferate locally and
differentiate to form a colony of donor cells, similar to
colony-forming unit-spleen (CFU-S). Differentiation of
mMAPCs into haematopoietic cells cannot be attributed to
contamination of the mMAPCs with HSC. BMMNCs are
depleted of CD45+ cells by column selection before MAPC
cultures are initiated. MAPCs are CD3, Gr-1, Mac-1, CD19,
CD34 and CD45 negative (Fig. 1), and early mesodermal or
haematopoietic transcription factors, including brachyury,
GATA-2 and GATA-1 (ref. 42), are not expressed in mMAPCs
(complementary DNA array data; not shown). Furthermore,
culture conditions for mMAPCs are not supportive for HSC and
all attempts at inducing haematopoietic differentiation from
hMAPCs in vitro have been unsuccessful to date28.

mMAPC engraftment was also seen in liver, intestine and lung
(Fig. 6). Because of the haematopoietic engraftment, we used
triple-colour immunohistochemistry to discriminate between
epithelial and haematopoietic cells in the same tissue sections.
In the liver, -gal+ CK18+ CD45- or -gal+ albumin+ cells
formed cords of hepatocytes occupying 5–10% of a given 5-µm
section surrounding portal tracts, a pattern seen in hepatic
regeneration from hepatic oval cells7, 15. The engraftment
pattern together with the finding that only 5 of 20 sections
contained donor cells, is consistent with the idea that stem cells
engraft in some but not all areas of the liver, where they
proliferate and differentiate into hepatocytes. Although CD45+
cells were identified in the same sections, no cell that stained
positive for both CD45 and CK18 was seen.

In the gut, crypts contain a population of 4–5 long-lived stem
cells5 that undergo several rounds of division in the middle and
upper portions of crypts, and give rise to epithelial cells that
migrate upwards, out of the crypt, onto adjacent villi.
Donor-derived -gal+ pan-CK+ CD45- epithelial cells entirely
covered several villi. In some villi, -gal+ pan-CK+ CD45- cells
constituted only 50% of the circumference (Fig. 6r, top
magnified panel), suggesting engraftment in one but not both
crypts. Multiple -gal+ pan-CK- cells were distinctly seen in the
core of intestinal villi (open arrow, Fig. 6q). These cells
co-stained for CD45 (Fig. 6r), indicating that they were
donor-derived haematopoietic cells. In the lung, approximately
4% of pan-CK+ CD45- alveolar epithelial cells were -gal+. A
number of recipient, pan-CK- CD45+ -gal- haematopoietic cells
can also be seen in the same section (Fig. 5t).

No contribution was seen to skeletal or cardiac muscle, tissues in
which, in contrast to epithelium, little or no cell turnover is seen
in the absence of tissue injury. Therefore, one may not expect
significant contribution of stem cells to these tissues. Although
mMAPCs differentiate into skin and tubular epithelium of the
kidney when introduced in the blastocyst, we did not find
engraftment in skin or kidney, in which epithelial cells undergo
rapid turnover. Despite the ability of MAPCs to differentiate
into neuroectoderm-like cells ex vivo, no significant engraftment
of mMAPCs was seen in the brain, and rare donor cells found in
the brain did not co-label with neuroectodermal markers. Two
recent publications demonstrated that donor-derived cells with
neuroectodermal characteristics were present in the brain of
animals that underwent bone marrow transplantation. However,
a fully ablative preparative regimen before transplantation or
transplantation in newborn animals was used19, 22, conditions
associated with breakdown of the blood–brain barrier. We
infused cells in non-irradiated adult animals, or animals treated
with a low dose of radiation, where the blood–brain barrier is
intact or only minimally damaged. This may explain the lack of
mMAPC engraftment in the central nervous system.

One animal developed a lymphoma in thymus and spleen after
16 weeks, as is commonly seen in aging NOD/SCID mice43.
Although the B-cell lymphoma was host-derived (CD19+ cells
were -gal-), approximately 40% of CD45- vWF+ cells in the
vasculature of the tumour stained with anti--gal antibodies,
indicating that mMAPCs can contribute functionally to
neoangiogenesis in vivo (Fig. 6k). Higher levels of mMAPC
engraftment and differentiation in radiosensitive organs—such
as the haematopoietic system and intestinal epithelium (Table 2,
P < 0.001)—after low-dose irradiation also suggests that
mMAPCs may contribute functionally to host tissues. Future
studies will be needed to show whether functional repopulation
occurs for other tissues in the post-natal transplantation setting.

We tested whether bone marrow from primary MAPC recipients
contained cells that engraft in secondary recipients. 1.5  107
bone marrow cells, recovered from primary recipients (Table 2:
animal 11 and 12) 11 weeks after mMAPC infusion, were
infused in secondary irradiated NOD/SCID recipients (Table 2,
animal SR-1 and SR-2). After 7 and 10 weeks, secondary
recipients were killed, and tissues were analysed for
engraftment. A similar pattern of engraftment was seen in
secondary recipients as in the primary recipients. Four to eight
per cent of bone marrow, spleen and peripheral blood cells were
 -gal+ CD45+; 6% and 8% of intestinal epithelial cells, and 4%
and 5% of lung epithelial cells were -gal+ pan-CK+ CD45-.
However, engraftment levels in the liver of secondary recipients
were lower than in the primary recipients (1% and 3% compared
with 5% and 8% -gal+ CK18+). This suggests that mMAPCs
may persist in the bone marrow of the primary recipient and
differentiate into haematopoietic cells as well as epithelial cells
when transferred to a second recipient.

As a control, we infused ROSA26 MAPCs grown to confluence
before injection. MAPCs allowed to become confluent lose their
ability to differentiate ex vivo in cells outside of the mesoderm,
and behave like classical mesenchymal stem cells28. Infusion of
106 confluent mMAPCs did not yield significant levels of donor
cell engraftment. Although few -gal+ cells were seen in bone
marrow, these cells did not co-label with anti-CD45 antibodies,
indicating that mesenchymal stem cells may engraft in tissues,
but are no longer able to differentiate into tissue-specific cells in
response to local cues.

In contrast with ES cells, we did not detect donor-derived
tumours in any of the animals. Although ES cells contribute to
all tissues when injected into the blastocyst, transplantation of
undifferentiated ES cells leads to the development of teratomas1,
which were not seen in our model. Furthermore, intravenous
infusion of ES cells does not usually lead to engraftment and
tissue-specific differentiation in vivo, unless ES cells have been
induced to commit to a lineage before transplantation.

Discussion
Three findings in our study address critically important
questions in the field of stem cell plasticity44. In vitro and in
vivo conversion of bone-marrow-derived MAPCs to
endothelium, ectoderm and endoderm occurs at the single cell
level. Second, the blastocyst studies indicate that MAPCs
contribute functionally to most somatic tissues. Third, robust,
early and persistent engraftment occurred in vivo when MAPCs
were transplanted into non-damaged recipients.

We found that MAPCs require culture conditions reminiscent of
ES cells, express at least some of the genetic markers of ES cells
(Oct-4, Rex-1, SSEA-1), have extensive proliferation and clonal
multilineage differentiation potential, contribute to all organs
when injected in a blastocyst, but engraft and differentiate into
tissue specific cells in response to organ-specific cues. Cell
fusion has recently been suggested as an explanation for stem
cell plasticity. In two studies somatic cells could be induced to
fuse with ES cells in vitro, generating tetraploid cells with
ES-like characteristics45, 46. Our in vitro studies demonstrating
that single euploid MAPCs—never co-cultured with
tissue-specific cells or ES cells—differentiate into cells of the
three germ layers, show that the in vitro behaviour of MAPCs
cannot be attributed to stem cell fusion. Although no definitive
studies were done in vivo to exclude the possibility that cell
fusion is responsible for the differentiation in multiple tissues,
the high frequency with which chimaerism was observed and the
balanced chimaerism differs from what was shown by ref. 46.
Finally, the speed and robustness with which engraftment and
tissue-specific differentiation is seen in animals without need for
selectable pressure, also argues against the idea that MAPC
engraftment and differentiation in post-natal animals is caused
by fusion.

A second possibility is that pluripotent stem cells persist even
after birth in multiple organs, and that when stimulated, they
proliferate and differentiate in response to local cues provided
by the organ they are recruited to. A third possibility is that a
tissue-specific stem cell may undergo genetic re-programming
in culture similar to that which occurs in the 'cloning process'47,
48. Monthly cytogenetics28 of mMAPCs and rMAPCs did not
reveal abnormalities, except in one mMAPC population that
became hyperdiploid at 45 population doublings, and was no
longer used for studies. When mMAPCs or rMAPCs were grown
to confluency, they stopped proliferating, and when cultured in
serum-free medium and differentiation-inducing cytokines after
40 or more than 120 population doublings, growth arrest and
terminal differentiation was seen. Furthermore, no tumour
formation was seen in immunodeficient mice that received
mMAPCs intravenously. Thus, even though re-programming
may have occurred in vitro under the MAPC culture conditions,
we have no evidence that transformation occurred.

Irrespective of their origin, MAPCs hold great promise for the
treatment of degenerative or inherited diseases. Similar to ES
cells, allogeneic MAPCs may be used to correct degenerative or
congenital diseases. MAPCs differentiate into haematopoietic
cells in vivo and can thus be used to establish haematopoietic
chimaerism, which should make such an allogeneic cell therapy
approach feasible. In contrast to ES cells, MAPCs can be
selected from autologous bone marrow and used undifferentiated
or after genetic manipulation in local or systemic therapies.
Furthermore, absence of teratoma formation when
undifferentiated MAPCs are infused should allow the use of
undifferentiated MAPCs to treat systemic diseases, such as
inherited enzyme deficiencies or muscular dystrophy.

Methods
Antibodies used Antibodies against NF-200 (clone N52, 1:400),
GalC (G-9152; 1:100), CK18 (C-8541; 1:300), pan-CK (C-2562;
1:100), albumin (A-6684; 1:100), GABA (A-2052, 1:500),
MAP2 (AP20, 1:400), dopa-decarboxylase (DDC-109, 1:100),
tyrosine hydroxylase (TH-16, 1:1,000), serotonin (S-5545,
1:1,000), cardiac troponin-I (sc-8118, 1:100), and dystrophin
(D-8168; 1:100) were from Sigma. Polyclonal antibodies against
Tau, vWF, Flk-1 and hepatocyte nuclear factor (HNF)-1 were
from Santa Cruz Biotechnology Inc. Antibody against GFAP was
from Santa Cruz Biotechnology or DAKO Corporation. Antibody
against CD31 was from BD PharMingen. Control mouse, rabbit
or goat immunoglobulin- and FITC, Cy3-labelled secondary
antibodies were from Sigma; Cy5-labelled antibodies were from
Chemicon International. Donkey anti-NeuN (1:100), anti-GFAP
(1:500), and anti--gal (1:2,000) were from Chemicon, Sigma
and Cortex Biochem, respectively. Secondary anti-donkey
antibodies (FITC, 1:200; Cy-3, 1:400; Cy-5, 1:200) were from
Jackson Immunoresearch. Anti--gal-FITC antibody was from
Rockland Immunochemicals. FITC- or phycoerythrin
(PE)-coupled antibodies against CD45, Gr-1, Mac-1, CD19,
CD3, CD13, c-Kit, Sca-1, CD34, Thy-1, Flk-1, MHC class I
(H–2Kk), MHC class II (I–Ak), and CD44 were from BD
Pharmingen, and anti-SSEA-1 antibody was from the
Developmental Studies Hybridoma Bank.

Differentiation culture and analysis Clonal eGFP+ or ROSA26
mMAPCs were replated in 60% DMEM-LG, 40% MCDB-201,
ITS, LA-BSA, 10-9 M dexamethasone, 10-4 M ascorbic acid
2-phosphate, 100 U penicillin and 1,000 U streptomycin28, 29.
Endothelial differentiation was induced with VEGF as
described28, 29. For neuroectodermal differentiation, 1  104
mMAPCs per cm2 were plated on FN with 100 ng ml-1 bFGF
(R&D Systems). Alternatively, cells were treated sequentially
with 100 ng ml-1 bFGF for 7 days, 10 ng ml-1 FGF-8 for 7 days
and 10 ng ml-1 BDNF for 7 days (R&D Systems). Hepatocyte
differentiation was induced as described30. Cells were fixed with
4% paraformaldehyde and methanol at room temperature, and
incubated sequentially for 30 min each with primary and
secondary antibodies. Between steps, slides were washed with
PBS/BSA. Cells were examined by confocal fluorescence
microscopy (Confocal 1024 microscope; Olympus AX70,
Olympus Optical Co. Ltd). To assess the frequency of different
cell types in a given culture, we counted the number of cells
staining positive with a given antibody in four low-power visual
fields (200–500 cells per field).

Tissue collection and analysis For the whole-mouse mount,
10-µm whole-body sections near the midline were prepared as
described41. Tissue sections were stained for -galactosidase
enzyme activity with the -gal staining kit from Invitrogen at pH
7.4. Manufacturer's instructions were followed except for
fixation, for which the tissue sections were incubated for 5 min
instead of 10 min.

A total of 0.5–1 ml of blood was obtained when animals were
killed, and bone marrow was collected by flushing femurs and
tibias. For phenotyping, red blood cells and bone marrow were
depleted using ice-cold ammonium chloride (Stem Cell
Technologies Inc.) and 105 cells were used for cytospin
centrifugation. For serial transplantation, 1.5  107 cells from
two femurs and two tibias were transplanted into individual
secondary recipients by means of tail vein injection. Cytospin
specimens of blood and bone marrow were fixed with acetone
for 10 min at room temperature.

Lungs were inflated with 1 ml 1:4 dilution of optimum cutting
temperature (OCT) compound (Sakura-Finetek Inc) in PBS.
Specimens of spleen, liver, lung, intestine, skeletal muscle,
myocardium, kidney and brain of the recipient animals were
collected and cryopreserved in OCT at -80 °C and in RNA Later
(Ambion Inc.) at -20 °C for Q-PCR. Five-micrometre-thick fresh
frozen sections were mounted on glass slides and fixed in
acetone for 10 min at room temperature. After incubation with
isotype sera for 20 min, slides were stained sequentially with
antibodies against cell-type antigens, anti--gal antibody, in
some instances antibodies against CD45, or a nuclear counter
stain (4,6-diamidino-2-phenylindole (DAPI) or TO-PRO-3).

Supplementary information accompanies this paper.

Received 30 January 2002;accepted 21 May 2002
 
 

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Acknowledgements. The authors wish to thank M. Jenkins for
technical support. This work was supported by NIH grants, the
Michael J. Fox Foundation, the Children's Cancer Research
Fund, the Tulloch Family Foundation, and the McKnight
Foundation. R.E.S., C.D.K., X.R.O.-G. and M.R. are supported
by the NIH-MSTP programme at the University of Minnesota.

Competing interests statement. The authors declare competing
financial interests.
 
 

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England.