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Pluripotency of mesenchymal stem cells derived from adult marrow

TL;DR: It is reported 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 meschymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro.
Abstract: 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.

Summary (2 min read)

5 Experimental Studies

  • The proposed technique is compared with baseline techniques over eight different image quality databases.
  • Each feature extraction scheme is followed by a feature-pooling phase that is carried out by using SVR.
  • The quality scores obtained for each feature extraction scheme and image quality database are compared with three correlationbased measures.
  • In Sections 5.1 and 5.2, the experimental procedures are explained in detail.

5.1 Image Quality Databases

  • Basic properties of each dataset, namely, number of reference images, number of distorted images, number of distortion types, number of distortion levels, image properties, and information about subjective evaluation are listed in Table 1 .
  • Some of the attributes in Table 1 are stated as "Varying" since these attributes may change for different reference images.

5.2 Experimental Procedures and Results

  • The average, standard deviation, and average ranking of the performance of the objective quality metrics.
  • In order to increase the readability of the results, objective quality measures providing the first and second best performance, are written in bold font.

Averaging scheme Criteria

  • Single distortion-based quality evaluation is performed on the LIVE image dataset, because the objective quality metrics that provide the leading performance on the overall quality evaluation (IFS, VSI, 2-DMC, and proposed) suffer performance losses on the LIVE image dataset.
  • Except for the results obtained for WN and GB, the VIF outperforms other metrics on the remaining distortion types.
  • GB, and FF types of degradation, it fails to provide acceptable performance on the compression type of degradation (JPEG2000, JPEG).
  • Taking into consideration the overall quality evaluations presented in Tables 2 and 3 , one can conclude that slight performance losses in single distortion results lead to performance reduction on the overall quality evaluation.
  • The experiments also revealed that the proposed 2-D complex mel-cepstrum outperforms classical 2-DMC features in nearly all of the tests by making use of image-phase information, which contains structural details and high-frequency components.

6 Conclusion

  • The proposed feature extraction framework integrates image-phase information with the classical mel-cepstrum computation to achieve an appropriate balance between FT magnitude and phase for image representation.
  • The complex mel-cepstrum-based features are fed into the SVR-based feature-pooling technique to obtain objective quality scores.
  • The experimental studies demonstrate that the proposed feature extraction technique outperforms baseline techniques on several datasets while achieving an acceptable performance on the remaining datasets.
  • The promising results obtained through large-scale experimentation reveal the effectiveness and representative power of the 2-D complex mel-cepstrum feature extraction scheme.

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Pluripotency of mesenchymal stem cells
derived from adult marrow
Yuehua Jiang*†, Balkrishna N. Jahagirdar*†‡, R. Lee Reinhardt§, Robert E. Schwartz*, C. Dirk Keenek, Xilma R. Ortiz-Gonzalezk,
Morayma Reyes*, Todd Lenvik*, Troy Lund*, Mark Blackstad*, Jingbo Du*, Sara Aldrich*, Aaron Lisberg*, Walter C. Lowk,
David A. Largaespada{ & Catherine M. Verfaillie*‡
* Stem Cell Institute, Division of Hematology, Oncology and Transplantation, Department of Medicine, § Department of Microbiology, Center for
Immunology, k Department of Neurosurgery, and { Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis,
Minnesota 55455, USA
These authors contributed equally to this work
...........................................................................................................................................................................................................................
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 humans
1
, their use in research as well as
therapeutics is encumbered by ethical considerations
2
. Stem cells
also exist for most tissues, including haematopoietic
3
,neural
4
,
gastrointestinal
5
, epidermal
6
,hepatic
7
and mesenchymal stem
cells
8
. 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
myoblasts
9,10
, cardiac myoblasts
11,12
, endothelium
11–14
, hepatic and
biliary duct epithelium
15–17
, lung, gut and skin epithelia
18
, and
neuroectodermal cells of donor origin have been detected
19–22
.
Some but not other studies demonstrated that neural stem
cells
23,24
as well as muscle cells
25,26
may differentiate into haemato-
poietic cells. When injected into a blastocyst, neural stem cells
contribute to a number of tissues of the chimaeric mouse embryo
27
;
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 mesench-
ymal stem cell cultures that can be expanded for more than 80
population doublings. This cell differentiates not only into
mesenchymal lineage cells but also endothelium
28,29
and endo-
derm
30
. 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)MAPCs
28
, 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 hMAPC
28
but different from that of murine
haemopoietic stem cells with ‘transdifferentiation potential
11,17,18,25
.
mMAPCs were 8–10
m
m 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).
Similar results were obtained when we isolated and cultured
MAPCs from bone marrow of Sprague–Dawley rats (n ¼ 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 (Supplemen-
tary 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-independent
31
whereas murine ES cells
require LIF for ex vivo maintenance
32
. Quantitative reverse tran-
scription 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).
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In vitro differentiation of single mMAPCs and rMAPCs
Approximately 1% of wells seeded with ten CD45
2
TER119
2
bone
marrow monocular cells (BMMNCs) yielded continuous growing
cultures, suggesting that 1 out of 1,000 CD45
2
TER119
2
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
þ
) 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 populations
35,36
. We sequenced
the genomic flanking region 3
0
from the retrovirus using splinker-
ette PCR, which detects a single retroviral insert in as few as 5 £ 10
3
cells
37
. 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
0
of the
insertion site.
We next tested the in vitro differentiation ability of mMAPCs and
0 100 200 300 400
Days in culture
Cell number
10
0
10
4
10
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36
a
c
d
b
CD45 Gr-1 Mac-1
CD19 CD3 CD13
c-Kit SSEA-1 Sca-1
CD34 Thy-1 Flk-1
MHC II MHC I CD44
FL1 height
10
0
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70
60
50
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0
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0
70
60
50
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0
Counts
70
80
60
50
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30
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10
0
23 kb
40 102 (PDs)
1234
1234
1234
Figure 1 Characteristics of mMAPCs. Mouse BMMNCs were plated with EGF, PDFG-BB,
and LIF on FN-coated plates. After 3–4 weeks, bead-selected CD45
2
/TER119
2
cells
were plated at ten cells per well in FN-coated 96-well plates in the same medium, and
expanded at 0.5–1.5 £ 10
3
cm
22
. a, Cells were enumerated at each passage under a
haemocytometer. b, mMAPCs cultured for 120 population doublings were labelled with
FITC-coupled antibodies against CD45, Gr-1, Mac-1, CD19, CD3, CD13, c-Kit, SSEA-1,
Sca-1, CD34, Thy-1, Flk-1, MHC class II (I–A
k
), MHC class I (H–2K
k
), CD44 or
immunoglobulin isotype control antibodies. Cells were analysed using a FACS-Calibur.
Black line, control immunoglobulin; red line, specific antibody. c, Telomere length of
mMAPCs cultured for 40 population doublings (PDs) (lane 1) and 102 population
doublings (lane 2)
28
. d, Quantitative RT–PCR for Rex-1 (left), Oct-4 (middle) and
b
-actin
(right). Lane 1, ladder; lane 2, ES cells; lane 3, MAPCs; lane 4, no-template control (see
Methods and Supplementary Information Table 1).
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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 £ 10
4
cells cm
22
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 dou-
blings). 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
þ
mMAPCs (Fig. 3) and clonal
eGFP
þ
rMAPCs (Supplementary Information Fig. 3).
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
21
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
þ
‘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
21
bFGF
38
.
After 14 days, 15 ^ 4% of MAPCs acquired morphologic and
phenotypic characteristics of astrocytes (glial acidic fibrillary pro-
tein (GFAP)
þ
), 12 ^ 3% of oligodendrocytes (galactocerebroside
(GalC)
þ
) and 68 ^ 9% of neurons (neurofilament-200 (NF-
200)
þ
). NF-200, GFAP or GalC were never found in the same cell.
More than 90% of eGFP
þ
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 mMAPCs
39
. When mMAPCs were
cultured sequentially with 100 ng ml
21
bFGF, 10 ng ml
21
FGF-8 and
finally 10 ng ml
21
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
g
-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 (Sup-
plementary Information Fig. 3) differentiate to endodermal cells. As
described more extensively elsewhere
30
, when replated on matrigel
with 10 ng ml
21
FGF-4 and 20 ng ml
21
hepatocyte growth factor
(HGF), approximately 60% of mMAPCs (Fig. 3a–h) or rMAPCs
acquired epithelioid morphology and 10% of cells became bi-
nucleated. 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
þ
. We recently showed
that these epithelioid cells have functional characteristics of hepato-
cytes, including urea and albumin production, phenobarbital-
inducible p450, gluconeogenesis and low-density lipoprotein
uptake
30
.
Southern blot analyses and flanking region PCR demonstrated
that differentiated eGFP
þ
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
þ
(Fig. 3a–h) and only a single retro-
viral 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 trans-
ferred 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/
b
-Gal
40
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
7 kb
6 kb
12 123 12345678
ab c
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). Mouse or
rat BMMNCs, transduced with an MSCV-eGFP retrovirus
49
, were depleted of CD45
þ
TER119
þ
cells, and eGFP
þ
cells were replated at ten cells per well and expanded for
more than 100 population doublings. DNA from MAPCs or their differentiated progeny
(Fig. 3 and Supplementary Information Fig.3) was digested overnight with Eco RI or Bam HI
(cut only once in the MSCV-eGFP provirus), fragments were separated by electrophoresis,
and probed with a
32
P-labelled eGFP cDNA probe. a, DNA extracted from pooled mMAPCs
differentiated after 80 population doublings into endothelial, neuroectodermal and hepatic
cells (see Fig. 3). A single retroviral insert at 7 kb was seen. b, Undifferentiated rMAPCs
(lane 1) and seven subpopulations of rMAPCs after 75 population doublings, subcultured
at 100 cells and expanded for 20 population doublings (lanes 2–8). A single retroviral
insert at 6 kb was seen. c, rMAPCs expanded for 85 population doublings differentiated to
the endothelial (lane 1), neuroectodermal (lane 2) and hepatic (lane 3) cells. A single
retroviral insert at 6 kb was seen.
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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 described
41
, and
stained with 5-bromo-4-chloro-3-indolyl-
b
-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.
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-
b
-gal fluorescein isothiocyanate (FITC) antibody (Fig. 5). Chimae-
ric animals that had Neo/
b
-Gal
þ
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, myo-
cardium, 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).
b
-gal
þ
cells
expressed markers typical for the tissue in which they had incor-
porated.
b
-gal
þ
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 immuno-
fluorescence to assure that
b
-gal
þ
cells in solid organs were not mere
haematopoietic cells.
b
-gal
þ
cells co-stained for pan-CK in the lung
and intestine, and for CK18 in the liver (Fig. 5). We also detected
CD45
þ
CK
2
b
-Gal
þ
cells in these organs. However, no cell co-
stained for all three antigens (that is,
b
-gal, CD45 and CK).
b
-gal
þ
cells co-stained for dystrophin in skeletal muscle and cardiac
troponin-I in the myocardium (Fig. 5).
b
-gal
þ
cells gave rise to
neurons (Neu-N
þ
) and astrocytes (GFAP
þ
) throughout the entire
brain, including the cortex, striatum, hippocampus, thalamus and
cerebellum (Fig. 5). In the cingulate cortex,
b
-gal
þ
neurons and
astrocytes were present, whereas in the underlying corpus callosum,
b
-gal
þ
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
b
-gal
þ
, and were interspersed with
b
-gal
þ
, MAPC-derived astrocytes.
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
embryo
27
. 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
þ
cells were seen in gonads, we have not yet
performed breeding experiments to test whether MAPCs contribute
to the germ line.
Figure 3 In vitro differentiation of mMAPCs to endothelium, neuroectoderm and
endoderm. ah, Clonal eGFP
þ
mMAPCs treated with VEGF (ac), FGF-4 þ HGF (df)or
bFGF (g, h) for 14 days. Cultures were stained with anti-vWF labelled with Cy3 (b), anti-
albumin-Cy3 (e), anti-GFAP-Cy5 (g) or anti-NF200-Cy3 (h). c, f, g and h show an overlay
of Cy3 or Cy5 staining with eGFP. a, c, d, f, g and h show that 100% of cells were eGFP
positive ( £ 10 magnification). ik, eGFP
þ
mMAPCs treated with VEGF for 14 days were
stained with antibodies against CD31-Cy3 (i), Flk-1-Cy3 (j) or vWF-Cy3 (k)(£ 60
magnification). lo, ROSA26 mMAPCs treated with bFGF for 14 days were stained with
antibodies against GFAP-Cy3, FITC-labelled NF200 and anti-GalC-Cy5 (l)(£ 20
magnification). Alternatively, ROSA26 mMAPCs treated sequentially with bFGF, FGF-8
and BDNF were stained with antibodies against MAP2-Cy3 and FITC-labelled anti-Tau
(m), GABA-Cy3 and FITC-labelled DDC (n), or TH-Cy3 and FITC-labelled serotonin (o)
( £ 40 magnification). p, q, eGFP
þ
mMAPCs treated with FGF-4 and HGF for 14 days
stained with antibodies against CK18-Cy3 (p), or albumin-Cy3 and HNF1-Cy5 (q)(£ 60
magnification). Colour staining: Cy3, red; FITC, green; Cy5, blue. Double positive for FITC
and Cy3, yellow. In q, the nucleus area is positive for HNP1-Cy5 and weakly positive for
Cy3, giving purple. Magnification: ah, £ 10; ik, £ 60; l, £ 20; mo, £ 40;
p, q, £ 60.
articles
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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
b
-gal/Neo-containing cells was tested by immunohistochemistry
(for
b
-gal) and Q-PCR (for Neo), 4–24 weeks after transplantation
(Table 2 and Fig. 6). Engraftment, defined as more than 1%
b
-gal
þ
cells by immunofluorescence and/or Q-PCR, was seen in haema-
topoietic 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.
b
-gal
þ
cells in bone marrow and spleen co-stained for CD45
(Fig. 6). Thirty-eight to 62% of bone marrow
b
-gal
þ
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
b
-gal
þ
CD3
þ
T cells were seen in
blood, bone marrow or spleen even though
b
-gal
þ
CD3
þ
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). Differ-
entiation of mMAPCs into haematopoietic cells cannot be attrib-
uted to contamination of the mMAPCs with HSC. BMMNCs are
depleted of CD45
þ
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 tran-
scription 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 haematopoie-
Table 1 Degree of chimaerism after ROSA26 MAPC injection
MAPCs per blastocyst Litters born No. of pups born Neo-positive by Q-PCR (%)
0 1–10 10–20 20–40 .40
...................................................................................................................................................................................................................................................................................................................................................................
10–12 4 of 11 22 5 of 22 (23%) 13 of 22 (59%) 2 of 22 (9%) 1 of 22 (4.5%) 1 of 22 (4.5%)
1 3 of 5 15 8 of 15 (53%) 5 of 15 (33%) 0 of 15 (0%) 0 of 15 (0%) 2 of 15 (13%)
...................................................................................................................................................................................................................................................................................................................................................................
One or 10–12 ROSA26 MAPCs were microinjected into 116 and 38 3.5-day-old C57BL/6 blastocysts, respectively. Blastocysts were transferred to 16 foster mothers and mice were allowed to develop and
be born: seven litters were born. The number of mice per litter varied between 1 and 8. After 4 weeks, chimaerism was assessed by comparing levels of Neo/
b
-gal in tail clippings of 4-week-old animals with
that of tissue from ROSA26 mice using Q-PCR. DNA, prepared by standard methods, underwent 40 rounds of amplification as described in Fig. 1. Primers used were Neo: 5
0
-TGGATTGCACGCAGGTTCT-
3
0
and 5
0
-TTCGCTTGGTGGTCGAATG-3
0
. Per cent chimaerism was determined by comparing the number of Neo copies in tail samples with that in tissue from ROSA26 mice according to manufacturer’s
recommendations (7700 ABI PRISM Detector Software 1.6).
Table 2 Engraftment levels in NOD/SCID mice transplanted with ROSA26 MAPCs
Animal Time Radiation Engraftment levels (%)
(weeks) Marrow Blood Spleen Liver Lung Intestine
.............................................................................................................................................................................
1 4 No 2 (1) 2 5 7 4 2
2 5 No 3 (4) 4 5 9 5 3
310No133632
4 16 No 4 2 3 4 3 4 (4.9)
524No323641
6 8 Yes 8 (8) 6 4 5 2 (1.1) 7
7 8 Yes 10 8 7 (7.3) 4 6 8
8 8 Yes 5 8 3 5 5 6
9 8 Yes 7 5 5 6 4 6
10 10 Yes 5 (6) 7 9 (12.5) 5 2 8
11 11 Yes 8 8 6 5 3 10 (11.9)
12 11 Yes 6 5 4 8 (6.2) 10 (12.3) 8
SR-1 7 Yes 6 7 5 1 (1.7) 5 8
SR-2 10 Yes 5 4 8 3 4 6
.............................................................................................................................................................................
Engraftment levels were determined by immunofluorescence or Q-PCR. 10
6
ROSA26 MAPCs were
injected intravenously in 6–8-week-old NOD/SCID mice, maintained according to IACUC guide-
lines. Recipient mice were either non-irradiated or irradiated with 250 cGy (57 cGy min
21
by a mark 1
caesium irradiator) 16–24 before cell infusion. After 4–24 weeks, animals were killed and organs
were evaluated by Q-PCR for Neo (in parenthesis) and immunohistochemistry (see Fig. 6). For SR-1
and SR-2, bone marrow from animals 9 and 10 was collected, and 1.5 £ 10
7
cells transplanted in
secondary recipients.
Figure 4 Chimaerism detection by X-gal staining and anti-
b
-gal staining in animals
generated from blastocysts microinjected with a single ROSA26 MAPC (see also Table 1
and Supplementary Information Fig. 4). ah, Images from X-gal-stained individual organs
from a 45% chimaeric mouse, determined by Q-PCR for Neo on tail clip. Tissue sections
were from: brain (a), skin (b), skeletal muscle (c), myocardium (d), liver (e), small intestine
(f), kidney (g) and spleen (h). i, j, Images from an X-gal-stained section through a mouse
that was not chimaeric (i) or was 45% chimaeric (j). Magnification, £ 20.
articles
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© 2002
Nature
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Group

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References
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Journal ArticleDOI
02 Apr 1999-Science
TL;DR: Adult stem cells isolated from marrow aspirates of volunteer donors could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages.
Abstract: Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

20,479 citations


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Journal ArticleDOI
06 Nov 1998-Science
TL;DR: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages.
Abstract: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

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05 Apr 2001-Nature
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Abstract: Myocardial infarction leads to loss of tissue and impairment of cardiac performance The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time1 Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation2,3,4,5; these events promote structural and functional repair6,7,8 This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein9 by fluorescence-activated cell sorting on the basis of c-kit expression10 Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells The developing tissue comprised proliferating myocytes and vascular structures Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease

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Journal ArticleDOI
27 Feb 1997-Nature
TL;DR: The birth of lambs from differentiated fetal and adult cells confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term and reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
Abstract: Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

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Frequently Asked Questions (14)
Q1. What contributions have the authors mentioned in the paper "Pluripotency of mesenchymal stem cells derived from adult marrow" ?

The authors 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. 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. 

Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strainleads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. 

In vitro and in vivo conversion of bone-marrow-derived MAPCs to endothelium, ectoderm and endoderm occurs at the single cell level. 

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. 

In two studies somatic cells could be induced to fuse with ES cells in vitro, generating tetraploid cells with ES-like characteristics45,46. 

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. 

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. 

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. 

Control mouse, rabbit or goat immunoglobuling and FITC, Cy3-labelled secondary antibodies were from Sigma; Cy5-labelled antibodies were from Chemicon International. 

Their 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. 

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. 

Bone marrow origin of endothelial progenitor cells responsible for postnatalvasculogenesis in physiological and pathological neovascularization. 

AAntibodies 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. 

C. M., Benveniste, P., Iscove, N. N. & van der Kooy, D. Hematopoietic competence is a rareproperty of neural stem cells that may depend on genetic and epigenetic alterations.