SUPPLEMENTAL INFORMATION
SI-Table 1: Selection and culture of murine MAPC
Cell population
Culture
condition
Cell
expansio
n
Phenotype*
Tri-lineage
differentiatio
n
Oct-4
CD45-Ter119- fresh cells
N=3
No LIF
FN/EGF/PDGF
None
ND
ND
ND
BMMNC depleted week
3-4 of CD45+Ter119+
cells
N=2
N=2
No LIF
6-8 weeks
6-8 weeks
ND
ND
ND
ND
ND
ND
6-8 weeks
ND
ND
ND
6-8 weeks
ND
ND
ND
FN/EGF/PDGF/LIF
>120PDs
yes
yes
C67BL/6: N=2
Laminin/EGF/PDG
F
ND
ND
C67BL/6: N=2
Collagen/EGF/PDG
F
ND
ND
C67BL/6: N=2
Matrigel/EGF/PDG
F
Stopped
after
30PDs
Stopped
after
30PDs
Stopped
after
30PDs
CD44,
MHC-I
negative
CD44,
MHC-I
positive
CD44,
MHC-I
positive
CD44,
MHC-I
positive
C67BL/6: N=2
FN/EGF/PDGF/LIF
+10% FCS
No
FN/EGF/PDGF/LIF
Density >2x103/cm4
CD44,
MHC-I
positive
CD44,
MHC-I
positive
No
C67BL/6: N=2
Stopped
after
30PDs
Stopped
after
30PDs
No
No
N=2
N=2
BMMNC depleted week
3-4 of CD45+Ter119+
cells
C67BL/6: N=3
ROSA26: N=3
*: See also SI-Fig.1
FN/EGF/PDGF
Laminin/EGF/PD
GF
Collagen/EGF/PD
GF
Matrigel/EGF/PD
GF
+ LIF
ND: Not done
Legend:
We have recently demonstrated that human post-natal bone BM contains primitive
progenitors that co-purify with mesenchymal stem cells, we have termed multipotent
adult progenitor cells, or MAPC. Human MAPC cultures were developed by testing
multiple conditions, including culture of BMMNC on fibronectin (FN), collagen-type-IV or
laminin. Media conditions tested included several concentrations of fetal calf serum
(FCS), epidermal growth factor (EGF), platelet derived growth factor (PDGF)-BB, acidic
fibroblast growth factor (aFGF), basic (b)FGF, bone morphogenetic protein (BMP)4,
insulin-like growth factor (IGF)1, and dexamethasone. Only when human CD45 and
GlyA depleted human BM mononuclear cells (MNC) were cultured with 0-2% FCS and
10 ng/mL EGF and 10 ng/mL PDGF-BB did we see growth of MAPC, that can be
cultured without senescence and differentiate into mesenchymal cells, endothelial cells
and hepatocyte like cells1-3 .
To isolate and culture expand MAPC from murine BM, we initially used methods
identical to those used for human MAPC. BMMNC from C57BL/6 (n=2; age 8 weeks)
were depleted of CD45 and TER119 positive cells using micromagnetic beads (Miltenyi
Biotec, Sunnyvale, CA) yielding <0.1% of BMMNC. CD45-TER119- cells were plated in
medium consisting of 60% DMEM-LG (Gibco-BRL, Grand Island, NY), 40% MCDB-201
(Sigma Chemical Co, St. Louis, MO), with 1X insulin-transferrin-selenium (ITS), 1X
lenolenic-acid-bovine-serum-albumin (LA-BSA), 10-9M dexamethasone (Sigma) and 10–
4
M ascorbic acid 2-phosphate (Sigma), 100U penicillin and 1000U streptomycin (Gibco)
on fibronectin (FN) (Sigma) with 2% fetal calf serum (FCS) (Hyclone Laboratories,
Logan, UT) and with epidermal growth factor (EGF) and platelet derived growth factor
(PDGF)-BB (both from R&D Systems, Minneapolis, MN). However, no MAPC could be
grown under these conditions.
We next tested whether we could culture mouse MAPC by initially plating BMMNC and
depleting hematopoietic cells ~3 weeks after initiation of the cultures. BM was obtained
from C57BL/6 animals (n=2, age 8 weeks). 105 BMMNC / cm2 were plated with 1-25
ng/mL EGF and 1-25 ng/mL PDGF-BB in FN, laminin, collagen type IV or matrigel (all
from Sigma) coated culture vessels. More than 80% of cells, presumed hematopoietic
cells, died during the first week. Cells were maintained between 2 and 5x103 cells/cm2.
After ~3 weeks, CD45-/Ter119- cells were selected (~20% of all cells) and subcultured at
10 cells / well. Cells were maintained between 0.5 and 1.5x103 cells/cm2 by sub-culturing
every 2-3 days at a 1:2 dilution. However, no long-term cell expansion was seen.
Because murine ES cells are dependent on leukemia inhibitory factor (LIF)4, and we had
shown that human MAPC express mRNA for the LIF-receptor, we next tested whether
addition of LIF would better support cell growth. BM was obtained from 3 C57BL/6 mice
or 3 ROSA26 mice (age 6-8 weeks) and BMMNC were plated on FN, laminin, collagen
type IV and matrigel in expansion medium with 1-25 ng/mL each EGF, PDGF-BB and
LIF (R&D Systems). After ~3 weeks, remaining CD45-/Ter119- cells were selected
(~20% of all cells), cells subcultured at 10 cells/well, and maintained between 0.51.5x103 cells/cm2. MAPC cultured on FN with 10 ng/mL EGF, 10 ng/mL PDGF-BB and
10 ng/mL LIF were CD3, Gr-1, Mac-1, CD19, CD34, CD44, CD45, cKit, and major
histocompatibility (MHC) class-I and class-II negative; expressed low levels of Flk1 and
Sca1, and higher levels of CD13 and SSEA-15 (Fig.1) The MHC-class-I and CD44
negative phenotype of mMAPC is similar to what we have shown for hMAPC1.
Approximately 1% of wells seeded with 10 CD45-/TER119- cells yielded continuous
growing cultures.
Culture of CD45-/TER119- cells on FN with cytokine concentrations <5ng/mL did not
yield continuous growing cultures. Culture of CD45-/Ter119- cells with 25ng/mL EGF or
PDGF did not improve growth of cells. Although cell expansion during the initial 2-3
months was greater when cells were cultured on matrigel, laminin or collagen type IV,
cells had phenotypic characteristics of MSC: CD44 and MHC class-I positive6. (See also
SI-Fig.1).
When cells were cultured with >2%FCS, they became larger, expressed CD44 and
MHC-class I antigens, and could not be induced to differentiate to endothelium or
neuroectodermal or endodermal cell types. Likewise, when cells were maintained at
higher cell densities (i.e. >2x103 cells/cm2), they became larger, expressed CD44 and
MHC-class I and could not differentiate to endothelium, neuroectoderm or endoderm.
To evaluate Oct-4 and rex-1 expression, RNA was extracted from mouse MAPC (PD 75)
and ES cells. mRNA was reverse transcribed and cDNA underwent 40 rounds of
amplification (ABI PRISM 7700, Perkin Elmer Applied Biosystems) with the following
reaction conditions: 40 cycles of a two-step PCR (95oC for 15 sec., 60oC for 60 sec.)
after initial denaturation (95oC for 10 min.) with 2l of DNA solution, 1X TaqMan SYBR
Green
Universal
Mix
PCR
reaction
buffer.
Primers
for
Oct-4:
5’-
GAAGCGTTTCTCCCTGGATT-3’ and 5’-GTGTAGGATTGGGTGCGTT-3’; for Rex-1: 5’GAAGCGTTCTCCCTGGAATTTC-3’ and 5’-GTGTAGGATTGGGTGCGTTT-3’. mRNA
levels in mMAPC were normalized using -actin, and compared with levels in mouse ES
cells. Products were visualized by electrophoretic separation on 1% agarose gels and
ethidium bromide staining.
References:
1. Reyes, M. et al. Purification and ex vivo expansion of postnatal human marrow
mesodermal progenitor cells. Blood 98, 2615-25 (2001).
2. Reyes, M. et al. Origin of endothelial progenitors in human post-natal bone marrow. J
Clin Invest 109:337-46 (2002).
3. Schwartz, R. E. et al. Multipotent Adult Progenitor Cells from Bone Marrow
Differentiate into Functional Hepatocyte-like Cells. J Clin Invest In Press (2002).
4. Williams, R. L. et al. Myeloid leukaemia inhibitory factor maintains the developmental
potential of embryonic stem cells. Nature 336, 684-7 (1988).
5. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts.
Science 282, 1145-7 (1998).
6. Pittenger, M. F. et al. Multilineage potential of adult human mesenchymal stem cells.
Science 284, 143-147 (1999).
SI-Table 2: Single cell origin analysis:
The murine stem cell virus-enhanced green fluorescent protein (MSCV-eGFP) retroviral
plasmid (gift from Dr. Pear, U. of Pennsylvania)1 was co-transfected with the pCL-Ampho
packaging plasmid in 293 cells (gift from Dr. Haas, U. of California, San Diego) 2.
Retroviral supernatants were collected in MAPC medium. Mouse or rat BMMNC
transduced with MSCV-eGFP were subcultured at 10 cells/well, eGFP+ cells populations
culture expanded for > 100PDs. RMAPC were subcultured after 75PDs at 100 cells/well
and expanded for 20PDs. In addition rMAPC and mMAPC were induced to differentiate
to endothelium, neuroectoderm and endoderm (Fig.3 and SI-Fig.3).
Flanking Region Splinkerette PCR: DNA from 106 mMAPC or rMAPC was prepared from
cells by standard methods. 300 ng genomic DNA was digested with AscI, which cuts
only once in the MSCV-eGFP provirus, and a splinkerette linker was ligated to the 5’ end
3
. The two oligonucleotides used for the splinkerette linker were as follows:
aattTAGCGGCCGCTTGAATTtttttttgcaaaaa (the hairpin loop forming sequence is in
lower case and the upper case is the reverse complement of the second splinkerette
oligo),
and
agtgtgagtcacagtagtctcgcgttcgAATTAAGCGGCCGCTA,
(the
underlined
sequence is also the sequence of the linker-specific primer (LS Primer) used for the
PCR and RT steps). We used a 5’-biotin-T7 coupled primer that recognizes a sequence
in the eGFP gene (Biotin-ggccagtgaattgtaatacgactcactataggctggCACATGGTCCTGCTG
GAG TTCGTGA; under-lined portion shows the minimum promoter sequence needed for
efficient in vitro transcription and the upper case is the eGFP specific sequence) and LS
primer to amplify the flanking regions for 10 rounds using Advantage 2 polymerase
(Clontech Laboratories, Palo Alto, CA, USA). The biotin labeled amplified product was
captured using streptavidin-magnetic beads (Streptavidin Magnetic Particles; Roche)
and further amplified using the T7 RNA polymerase, approximately 1,000 fold, and then
DNAase 1 treated. The product was reversed transcribed using the LS primer according
to the superscript II protocol (Gibco-BRL, Grand Island, NY), and subsequently amplified
by 30 rounds of nested PCR using the primer for the 3’LTR. The flanking DNA in the
host genome was sequenced. The sensitivity of this technique is 1 target molecule in a
cell population of 5,000 cells1.
Confirmation by Flanking Region Specific PCR: Specific primers were generated to
amplify the genomic sequence between the 3’ MSCV-LTR and the host-flanking
genome. Quantitative PCR was used to quantify the relative amount of flanking
sequence compared with the eGFP sequence. Flanking sequences and primers are
shown in Table below.
Clone
Genomic sequence
Rat flanking sequence
GATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACC
TCGGGGGTCTTTCAAAGTAACTCCAAAAGAAGAATGGGTTGT
TAGTTATTAAACGGTTCTTAGTAAAGTTTTGGTTTTGGGAATC
ACAGTAACAACTCACATCACAACTCCAATCGTTCCGTGAAA
match in mouse genomic database = G10P608574FH4
Mouse flanking sequence
GATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCATA
AGTTATAAGCTGGCATGACTGTGTTGCTAAGGACACTGGTGA
AAGC
match in rat genomic database = TUWEP1E43505A
Bold: MSCV LTR
Bold and underlined: MSCV LTR primer used for Q-PC
Italics and underlined: Flanking sequence primers used for Q-PCR
1. Pear, W. S. et al. Efficient and rapid induction of a chronic myelogenous
leukemia-like myeloproliferative disease in mice receiving P210 bcr/abltransduced bone marrow. Blood 92, 3780-92 (1998).
2. Norris, P., Jepsen, K. & Haas, M. High-titer MSCV-based retrovirus generated in
the pCL acute virus packaging system confers sustained gene expression in
vivo. J Virol Methods 75, 161-167 (1998)
3. Lenvik, T., Lund, T. C. & Verfaillie, C. M. Blockerette-ligated catpture T7
amplified RT-PCR, a new method for determining flanking sequences. Molecular
Therapy In Press (2002).
SI-Table 3: Q-RT-PCR Otx1, Otx2, Pax2, Pax5 and nestin
otx12
otx22
Nestin2
Pax22
Pax52
5’-GCTGTTCGCAAAGACTCGCTAC-3’;
5’-ATGGCTCTGGCACTGATACGGATG-3’
5’-CCATGACCTATACTCAGGCTTCAGG-3’;
5’-GAAGCTCCATATCCCTGGGTGGAAAG-3’
5’-GGAGTGTCGCTTAGAGGTGC-3’;
5’-TCCAGAAAGCCAAGAGAAGC-3’
5'-CCAAAGTGGTGGACAAGATTGCC-3',
5'-GGGATAGGAAGGACGCTCAAAGAC-3'
5’-CAGATGTAGTCCGCCAAAGGATAG-3’,
5’-ATGCCACTGATGGAGTATGAGGAGCC-3’
RNA was extracted from mMAPC and neuroectodermal differentiated progeny (day 1- 7
after addition of bFGF), or mouse brain as described1. mRNA was reverse transcribed
and cDNA underwent 40 rounds of amplification by Q-PCR amplification as for NEO-QPCR. mRNA levels were normalized using GAPDH as housekeeping gene, and
compared with levels mouse brain tissue (nestin, Otx1, Otx2, Pax2, Pax5).
1. Reyes, M. et al. Purification and ex vivo expansion of postnatal human marrow
mesodermal progenitor cells. Blood 98, 2615-2625. (2001).
2. Lee, S. H., Lumelsky, N., Studer, L., Auerbach, J. M. & McKay, R. D. Efficient
generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat
Biotechnol 18, 675-679 (2000).
SI-Table 4: Hematopoietic engraftment in NOD/SCID mice transplanted with 106
ROSA26 MAPC
Animal #
Week
XRT
Total %
contribution
Gr-1
Mac-1
CD19
Ter119
% of total
#1
4
N
2
38
27
18
11
#5
24
N
3
62/70*
9/6*
26/7*
5/9*
#7
8
Y
10
47/60*
18/24*
20/10*
9/10*
#10
10
Y
5
56
12
31
4
Legend: 106 ROSA26 MAPC were injected via tail vein into 6-8-week old NOD/SCID
mice with or without 250cGy irradiation. Animals were sacrificed 4-24 weeks after MAPC
infusion. 0.5-1 ml of blood was obtained when animals were sacrificed and BM was
collected by flushing femurs and tibias. For phenotyping, red cells in blood and BM were
depleted using ice cold ammonium chloride and 105 cells used for cytospin
centrifugation. Slides were stained with anti--gal-FITC Ab and PE-conjugated Abs to
CD45, CD19, Mac-1, GR-1 and TER119. Slides were examined under fluorescence
microscope and the number of PE-positive cells per 100 -Gal positive and per 100 Gal negative cells counted.
* x/y:
x=percent -gal positive cells (donor cells) expressing the marker of interest
y=percent -gal negative cells (host cells) expressing the marker of interest
SUPPLEMENTAL INFORMATION FIGURES
SI-Figure 1: Morphology and FACS phenotype of MAPC
Mouse BMMNC were plated for 3-4 weeks on FN, collagen, laminin matrigel with 10
ng/mL EGF, PDGF-BB and LIF. CD45 and TER119 cells were depleted and CD45TER119- cells plated at 10 cells / well on the same ECM components and with the same
cytokines. Cells were sub-cultured every 36-48h to keep cell densities between 500 and
1,500/cm2.
Panel a: Light microscopy of MAPC after 55 population doublings
Panel b: MAPC cultured on fibronectin for 30PDs or 120PDs or on matrigel, collagen or
laminin for 30PDs were collected by trypsinization and stained with FITC- or PE-coupled
Abs against CD44, CD45, MHC class-I or IgG isotype control Abs. Cells were analyzed
using FACS-Calibur.
SI-Figure 2: rMAPC expansion and telomere length
BM was obtained from Sprague Dawley rats. Rat BMMNC were plated for 3-4 weeks on
FN, with 10 ng/mL EGF, PDGF-BB and LIF. CD45 and Ter119 cells were depleted and
CD45-Ter119- cells plated at 10 cells / well on the same ECM components and with the
same cytokines. Cells were sub-cultured every 36-48h to keep cell densities between
500 and 1,500/cm2. (See SI-Table 1 for further description of method).
Panel a: Cells were enumerated at each passage under hemocytometer.
Panel b: rMAPC that had undergone 40 to 100PDs were harvested and telomere length
evaluated (using methods previously described28). rMAPC after 42 PDs (lane 1), 75 PDs
(lane 2), 86 PDs (lane 3) and 100 PDs (lane 4).
SI-Figure 3. In vitro differentiation of rMAPC to endothelium, neuroectoderm and
endoderm
Clonal eGFP+ rMAPC were induced to differentiate to endothelium, neuroectoderm and
endoderm as described. After 14 days, cells were fixed, stained with Abs against lineage
specific antigens and examined by confocal microscopy.
a-b: VEGF-treated eGFP+ rMAPC stained with anti-CD31 (a), or vWF (b) / Cy3conjugated Ab.
c-e: bFGF treated eGFP+ rMAPC stained with anti-GFAP / Cy3-conjugated Ab combined
with an anti-NF200 / Cy5-conjugated Ab (c), anti-GalC / Cy3-conjugated Ab (d), anti-Tau
Cy3-conjugated Ab (e).
f-g: FGF4 and HGF treated eGFP+ rMAPC stained with anti-albumin / Cy3-conjugated
Ab and anti-HNF1 / Cy5-conjugated Ab (f) or anti-CK18 / Cy5-conjugated Ab and antiHNF1/ Cy3-conjugated Ab (g).
SI-Figure 4: X-gal staining to determine chimerism in mice generated from
blastocysts microinjected with ROSA26 MAPC.
X-gal staining of individual organs: brain (a), skin (b), skeletal muscle (c), heart muscle
(d), liver (e), gut (f), kidney (g) and spleen (h), harvested from blastocysts microinjected
mouse that had no donor cell chimerism (Panel I) and a blastocysts microinjected mouse
that had 7% donor cell chimerism (Panel II) by NEO PCR of tail clipping.