Human ESC and iPSC cultures. The hESC
lines used in this study were H1, H7, and H9
[24]; nine Harvard Stem Cell Institute hESC
lines [25] and thirteen hESC lines derived at
Advanced Cell Technology[26,27] (See Table1
and supplementary Table 1). The hiPSC lines
used were iPS(IMR90)-1, iPS(Foreskin)1-1,
iPS(Foreskin)4-1, and 4-3, which were generated
using Thomson’s four factors (Oct-4, Sox-2,
Nanog and Lin28) with Lentiviral vector [1]; rvhiPS01,
rv-hiPS02, rv-hiPS03 and rv-hiPS04,
which were derived by using Yamanaka’s four
factors (Oct-4, Sox-2, c-Myc and Klf4) with
retroviral vector [6]. Cells were cultured on
mitomycin-treated feeder MEFs in hESC
medium supplemented with 20% Knockout
Serum Replacement (Invitrogen), 10 ng/ml
bFGF (Stemgent), ��-mercaptoethanol (Sigma),
NEAA, GlutaMax-I and
penicillin/streptomycin(Invitrogen). Cells were
fed with fresh medium daily and confluent
hESC/hiPSC cultures were split using 1 mg/ml
collagenase IV in DMEM/F12 (Invitrogen).
Embryoid body (EBs) formation. hESC/hiPSC
cultures were incubated with collagenase IV at
37o C for up to 10 minutes, and the large clumps
of cells were collected. The clumps were washed
once with 5 ml hESC medium. To initiate
differentiation, the cell clumps were resuspended
in EB differentiation medium [Stemline II
(Sigma) supplemented with 50 ng/ml BMP4
(R&D System) and VEGF (Invitrogen)], at a
density of approximately 2× 106 cells per well of
a 6-well ultralow attachment plate (Corning).
Generation of hemangioblasts/BCs.
Hemangioblasts/BCs were generated as
previously described [21,22]. Briefly, EBs were
cultured in differentiation medium for 48 hours,
and then half of the medium replaced with
medium containing BMP4 (50 ng/ml), VEGF
(50 ng/ml) and bFGF (20 ng/ml). After 24-hour
incubation, the EBs were collected, washed once
in 2 ml D-PBS (Invitrogen), and incubated with
1 ml of trypsin/EDTA (Invitrogen) at 37o C for
2-3 minutes. The EBs were dissociated by
pipetting up and down 10 times using a 1000 μl
pipette or until the cell suspension became
homogenous with no visible clumps. An equal
volume of MEF medium was added and the cells
were centrifuged at 210g for 4 minutes. The cell
pellet was resuspended in Stemline II medium to
1-2 �� 106 cells/ml, 5 �� 104 cells plated in 1- 2 ml
of blast growth medium (BGM) [21,22], and
then incubated for up to 6 days.
Characterization of hematopoietic colony
forming capability. Day 6 hiPSC-BCs and
hESC-BCs were purified from blast cell cultures.
1 × 103 blast cells were resuspended in 20 ��l of
Stemline II medium, mixed well with 0.5 ml of
either CFC medium (Stem Cell Technologies,
H4436 only) or BGM (H4436 plus cytokines)
using 2 quick vortex pulses. Cells in semi-solid
CFU assay medium were plated into wells of a
24-well Ultra-low attachment plate for CFU
colony growth. Cells were incubated in a CO2
incubator at 37°C for up to 15 days. The
formation of hematopoietic colonies was
monitored microscopically. On day 15, the
number of hematopoietic colonies in each well
as well as their morphology was recorded. For
the CFU-MK assay, BCs were purified and
plated according to the manufacturer’s
instructions using a MK CFU assay kit (Stem
Cell Technologies, Cat 04973).
Differentiation of hiPSC-BCs and hESC-BCs
into erythroid cells. Erythroid differentiation
was carried out as previously described[23].
Briefly, day 5 blast cells from both hiPSCs and
hESCs were purified and plated into 24-well
plates (30,000 cells/ well) with BGM medium
and allow to expand for 5-7 day or until the
culture become confluent. At the end of each
expansion, cells were collected from each well
and counted. To expand further, equal numbers
of cells from both hiPSC-BCs and hESC-BCs
were replated in BGM medium. A total of three
expansion cycles were performed in this study
(each expansion experiment was carried out in
triplicate). To further differentiate toward
erythroid cells, expanded BGM medium with
cells was mixed at 1:1 ratio with Stemline II +
EPO (3U/ml, Cell Sciences) and cultured for an
additional 6-8 days. Additional Stemline II +
EPO medium was added every 2-3 days. The
erythrocytes were then spun onto glass slides and
stained for benzidine/Giemsa (Sigma) and
erythrocyte marker CD235a (DAKO) as
described previously [23].
Differentiation of endothelial cells from hiPSCBCs
and hESC-BCs. Purified BCs from both
hESCs and hiPSCs were plated into wells of
fibronectin-coated 24-well plate (BD
Biosciences) with EGM-2 medium (Lonza) at
the density of 36,000 cells per well (n=4).
Endothelial cells differentiated from hESCs and
hiPSCs were sub-cultured when they reached
confluence, and were continuously cultured for
up to 30 days. The morphology and growth rate
of endothelial cells were recorded. To test the
endothelial clonogenic capability of BCs derived
from hESCs and hiPSCs, individual blast
colonies were handpicked and plated for
endothelial cell colony formation.
Endothelial markers and cell senescence
staining. The BC-derived endothelial cells were
allowed to grow for 10 days in EGM-2 medium.
The endothelial cells derived from hiPSCs were
trypsinized and plated onto Matrigel (BD
Biosciences) for network structure formation as
described previously[21]. Expression of
endothelial markers CD31(DAKO), VE-Cad
(Cell Signaling Technology) and vWF (DAKO)
on the hiPSC-derived endothelial cells was
confirmed by immunofluorescence staining as
described previously[21]. LDL uptake of hiPSCendothelial
cells was evaluated by culturing the
cells in the presence of Alexa-Fluor 594 Ac-LDL
(Invitrogen) at 10 ��g/ml in EGM-2 for 8 hours,
followed by washing with medium 3 times. The
intracellular fluorescent LDL was examined
using fluorescence microscopy. For detection of
endothelial cell senescence, cells were fixed
between 20 to 30 day days after initiation of
endothelial differentiation and then stained for
endogenous ��-galactosidase using a Cell
Senescence Kit (Cell Signaling Technology)
according to the manufacturer’s manual.
