Supplementary Information
Generation of Human Epidermis-Derived Mesenchymal Stem Cell-like
Pluripotent Cells (hEMSCPCs)
Bing Huang1*, Kaijing Li1, Jie Yu1, Min Zhang1, Yongping Li1, Weihua Li1, Wencong Wang1 ,
Liping Guan1, Wenxin Zhang1, Shaochun Lin1, Xintao Huang1, Liping Lin1, Yongliang Lin1,
Yichi Zhang1, Xinming Song2, Zhichong Wang1, Jian Ge1*
1. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen
University,
GuangZhou 510060, China
2. Genetic Laboratory of Sun Yat-sen University, GuangZhou 510080, China
Supplementary Methods
PCR of Human SRY
To identify hEMSCPC-derived cells in the female offspring, genomic DNA was extracted
from chimera tissue samples using the Biomiga EZgene TM Tissue gDNA Miniprep Kit and
human genomic DNA detected using a two-step nested PCR procedure and primers specific
for the human SRY sequence: F1, 5′-CAGTGTGAAACGGGAGAAAACAGT-3′; R1, 5′CTTCCGACGAGGTCGATACTTATA-3′; F2, 5′TGTAATTTCTGTGCCTCCTGGAAGAATGG-3′; and R2, 5′GAAACGGGAGAAAACAGTAAAGGCAACGT-3′. In the first PCR reaction, human SRY
was amplified in a 25 μL PCR reaction volume using primers F1 and R1. The thermocycle
used was 95°C for 10 min, 35 cycles of 30 s at 95°C, 30 s at 65°C, and 30 s at 72°C, and a
single 10 min extension step at 72°C. The second PCR reaction amplified 0.5 μL of the first
PCR product in a 25 μL reaction volume containing F2 and R2. The same thermocycle
protocol was used except that the number of cycles was reduced to 32. Amplification of
GAPDH with primers F, 5′-TCACTCAAGATTGTCAGCAA-3′ and R, 5′AGATCCACGACGGACACATT-3′ was used as a control. The thermocycle for GAPDH PCR
expansion was an annealing temperature at 55°C for 27 cycles. The PCR products were
separated by 1.5% agarose gels.
PCR and Electrophoresis Reagents and Equipment
We used the Biomiga EZgene TM Tissue gDNA Miniprep Kit (Biomiga Inc., San Diego,
USA), 2× Taq PCR Mix (Biomiga Inc), Biowerter Regular Agarose G-10 (Gene Company
LTD., Hong Kong), and a TProfessional Standard Gradient Thermocycler (Biometra GmbH,
Germany). A DYY-12 (Beijing Liuyi Instrument Factory, China) with a Vilber Lourmat
Infinity gel formatter was used for electrophoresis (Vilber Lourmat, France). An ultraviolet
spectrophotometer was purchased from Beckman-Coulter (Model DV800; Beckman Coulter,
Inc. USA).
Fluorescence in situ Hybridization (FISH) for Detection of the Human Y Chromosome
in hEMSCPCs
When cultures were 70%−80% confluent, the medium was exchanged and 0.2 g/ml
colcemid added. Cultures were incubation at 36.8 ± 0.2°C under 5% CO2 and 100% humidity
for 2−2.5 hours, the medium containing colcemid removed, and cells rinsed with PBS (×2).
Cells were harvested, counted, rinsed once with PBS, and resuspended by vortex oscillation.
Approximate 2 × 106 cells were added to 3 mL of 0.075 M KCl, gently blended and incubated
at 37°C for 10 min and centrifuged at 1,200 rpm for 5 min. The supernatant was removed and
cells fixed in 3 mL of freshly prepared pure methanol and glacial acetic acid (3:1) under
gentle vortexing to prevent clumping. The centrifugation and resuspension steps were
repeated and the final suspension adjusted to 1×106 cells/mL(in the same fixative) . The
suspension was divided into microcentrifuge tubes, 100 µL per tube, and the tubes stored at
-20°C for subsequent use. Prior to FISH, cells were thawed to room temperature, and
cytospinning (Shandon, Thermo Electron Corporation, UK) was performed at 2,000 rpm with
moderate acceleration over 2 min. The resultant cell smears were air-dried, soaked in
2×SSC/0.1%NP-40 rinsing solution (prepared based on the regimen provided by Abbott
Molecular Inc. USA) preheated to 37°C for 30 min, and excess solution removed. Smears
were dehydrated by gradient ethanol (70%, 85%, and 100% for 2 min in each gradient) at
room temperature, and then air-dried. The following steps were performed under
light-shielding. A human Y chromosome-specific probe mixture (10 µL per smear, prepared
based on the regimen provided by Abbott Molecular Inc. USA) was added to the smears. The
smears were then covered with slips and subjected to co-denaturation for 6 to 7 min at 73 ±
1°C. For hybridization, smears were placed in a sealed humid container, preheated to 42°C,
for 16 hours. Following hybridization, smears were rinsed with prewarmed (73 ± 1°C)
2×SSC/0.3% NP-40 solution (according to instruction provided by Abbott Molecular Inc. USA)
for 2 min and then transferred to 2×SSC/0.1% NP-40 solution at room temperature for
another 2 min. Excess solution was removed, the smears air-dried, counterstained with 10 µL
DAPI solution (Abbott Molecular Inc. USA), and photographed 30 min later under a laser
confocal microscope (Zeiss, Germany) using blue light (405 nm excitation, 461 nm emission)
and red light (543 nm excitation, 588 nm emission).
FISH Detection of the Human Y Chromosome in Chimera Blood
Anti-coagulated blood was obtained from mice and 200 L added to 3 mL of 0.83% NH4Cl
solution pre-cooled to 4 °C. The blood/NH4Cl solution was gently pipetted, left to stand at
37°C for 15 min, mixed with 4 mL PBS, remixed, and centrifuged at 1,200 rpm for 5 min.
The supernatant was removed and the cells suspended in 10 mL PBS, mixed, and centrifuged
at 1,500 rpm for 5 min. These steps were repeated once more and the cell pellet suspended in
3 mL of 0.075 M KCl. All subsequent steps were as described for FISH for Detection of the
Human Y Chromosome in hEMSCPCs.
.
Immunofluorescence Staining and FISH of Chimera Blood
Immunofluorescence staining prior to FISH was performed in some cases using antibodies
that showed no cross-reactivity to wild type female mouse blood in pilot experiments. Briefly,
anticoagulated blood (200 L) was treated as described (FISH Detection of the Human Y
Chromosome in Chimera blood) and cells suspended in 100 µL PBS. Subsequent steps were
performed under light shielding. To the suspension was added an FITC-labeled anti-human
monoclonal CD3, CD19, or CD16 antibody (Invitrogen, USA, titer based on the formula
provided by the manufacturers). The suspension was gently pipetted, incubated at room
temperature for 30−40 min (according to the procedures provided by the reagent
manufacturers), rinsed in PBS, and centrifuged at 1,800 rpm for 5 min. The
rinse/centrifugation/resuspension steps were repeated and the final cell pellet suspended in 1
mL pure methanol and glacial acetic acid (3:1) under gently vortexing. After centrifugation at
2,000 rpm for 5 min, the supernatant was removed and another 100 µL of fixative added drop
by drop under gentle blending. Cytospinning was performed at 2,000 rpm with moderate
acceleration over 2 min. The smears were air-dried and then stained as described for FISH
Detection of the Human Y chromosome in hEMSCPCs. Stained smears were observed and
photographed under a laser confocal microscope (Zeiss, Germany) using blue light (405 nm
excitation, 461 emission), green light (488 nm excitation, 525 nm emission), and red light
(543 nm excitation, 588 nm emission).
Immunofluorescence Staining and FISH Detection of the Human Y Chromosome in
Chimera Brain, Heart, Lung, Spleen, Liver, and Kidney
No antibody used for combined immunofluorescence and FISH showed cross-reactivity in
wild type female mice. The antibodies used for indirect immunolabeling were a rabbit
anti-human β-III tubulin polyclonal antibody and a rabbit anti-human MAP2 monoclonal
antibody (Abcam, USA). Immunolabeling was visualized using an FITC-conjugated goat
anti-rabbit secondary antibody (SouthernBiotech, USA). Other antigens were labeled directly
with FITC-conjugated mouse or rabbit anti-human monoclonal antibodies against troponin I,
SP-C, CD31, CD16, ALB, or VEGF R2 (all antibodies from Invitrogen, USA, Santa Cruz,
USA, or BD, USA).
Brain, heart, lung, spleen, liver, and kidney tissues from female wild type and chimeric mice
were embedded in OCT compound (Sakura Finetek, USA) and stored at -20°C for subsequent
use. Tissues were tested for human Y chromosome expression by nested PCR and positive
frozen sections sliced at 4 µm. Slices were air-dried and OCT gel removed. To improve
adhesion and to expose structures to antibodies, smears were prepared as described. All
subsequent steps were performed under light shielding. Slices were incubated with
FITC-conjugated antibodies or primary antibodies for 40 min at room temperature according
to the procedures provided by reagent manufacturers, rinsed in PBS pre-cooled to 6−8°C (×3
or ×4). For indirect labeling, slices were incubated in an FITC-conjugated secondary antibody
at room temperature for 30 min and rinsed in PBS pre-cooled to 6−8°C (×3 or ×4). After
incubation with antibodies, tissue slices were air-dried and soaked in prewarmed (37°C)
2×SSC/0.1%NP-40 solution (prepared as recommended by Abbott Molecular Inc. USA) for
30 min. Excess solution was removed and the slices dehydrated in an ethanol gradient,
air-dried, and hybridized as described for hEMSCPCs. Hybridized tissue smears were
counterstained with 10−15 µL DAPI solution (Abbott Molecular Inc. USA) and photographed
under a laser confocal microscope (Zeiss, Germany) using the same blue, green, and red
excitation/emission wavelengths.
Counting tissue specific antibody-positive cells in human SRY gene-positive mouse blood
samples
We randomly selected five human SRY gene-positive mouse blood samples and calculated the
average fractions of cells expressing CD3, CD16, and CD19 from 5 fields per sample.
Supplementary Tables
Supplementary Table 1 Dynamic expression of specific markers in cultured hEMSCPCs
before passage 30 as determined by flow cytometry
Marker
P2
P10
P20
P30
P*
CD73
97.975±0.689
98.438±0.574
98.713±0.296
97.625±0.697
1.182
CD90**
62.025±11.563
91.863±1.387a
90.650±3.659b
94.825±2.015c
0.002
CD105
46.700±7.356
59.225±8.041
59.075±4.730
65.813±3.793
0.171
Vimentin
77.225±5.653
88.425±2.884
87.8±4.413
86.475±4.463
0.271
Nestin***
62.263±9.913
87.325±2.978
GFAP
0.325±0.269
1.3±0.88
β-III Tubulin
3.113±1.367
CD10
A
C
87.163±2.246
0.003
1±0.528
1.975±1.062
0.347
6.288±2.609
4.525±1.607
4.488±2.434
0.765
0.838±0.404
3.488±1.630
2.963±0.968
2.025±0.652
0.227
CK19
0.225±0.156
0.188±0.174
0.35±0.151
0.138±0.089
0.265
CD34
0.388±0.090
0.575±0.185
0.15±0.05
0.413±0.146
0.138
CD45
0.05±0.038
0.025±0.025
0.038±0.018
0.15±0.07
0.134
CD3
0.375±0.096
0.4±0.08
0.25±0.06
0.763±0.293
0.093
CD19
0.125±0.059
0.063±0.042
0.063±0.026
0.1±0.033
0.649
CD16
0.1±0.027
0.038±0.018
0.163±0.068
0.175±0.077
0.116
CD31
0
0.513±0.309
0.425±0.256
0.438±0.180
0.241
VEGFR2
0
0.7±0.411
0.538±0.263
0.7±0.211
0.139
HLA-DR
4.438±3.784
0.05±0.032
0.038±0.026
0
0.284
12.113±7.633
6.95±5.576#
8.688±5.151
0.023
HLA-I****
27.563±10.104
92.875±1.627
B
* SPSS 13.0 was used for statistical analysis. Data from FACS are presented as mean ± standard error of
the mean (SEM). Results were analyzed by two-way ANOVA with Bonferroni multiple comparison
post-tests for pair-wise comparisons. Differences were considered statistically significant when P < 0.05.
Data from n=8 independent cultures.
** hEMSCPCs at passage 2 had lower CD90 expression compared to passages 10, 20, and 30. a The
comparison between passage 2 and passage 10, P= 0.009, b The comparison between passage 2 and
passage 20, P= 0.013, c The comparison between passage 2 and passage 30, P=0.004.
*** hEMSCPCs at passage 2 had lower nestin expression compared to passages 10, 20, and 30. A P= 0.021,
B P= 0.004, C P= 0.022.
**** hEMSCPCs at passage 2 had higher HLA-I expression compared to passage 20, #P= 0.035.
Supplementary Table 2 Organs or tissue harboring cells derived from hEMSCPCs as
revealed by nested PCR of human SRY genes and immunofluorescence staining of
tissue-specific antibodies
Number
Blood
Heart
6
8
+
10
+
11
+
13
+
15
+
17
+
18
Brain
Lung
Kidney
+
+
+
+
19
+
+
20
+
+
21
+
22
+
+
+
+
32
+
34
+
36
+
+
37
+
+
+
+
+
+
38
+
+
41
+
42
+
45
+
+
50
52
+
+
53
63
+
+
+
+
64
66
Liver
+
24
39
Spleen
+
+
Neither wild type mice (numbers 1 to 5) nor mice with 7 negative organs are shown.
hEMSCPCs were most widely distributed in blood, followed by lung, heart, brain, spleen,
kidney, and finally liver (the lowest).
Supplementary Table 3 Tissue specific antibody-positive cells in 5 human SRY
gene-positive mouse blood samples
Tissue-specific
antibody
Positive cell numbers/Total cell numbers
Positive rate (%)
1
2
3
4
5
The average
number ± SD (%)
CD3
24/118
20.338
11/76
14.474
16/54
29.630
10/57
17.544
16/59
27.119
21.821±6.394
CD16
8/49
16.327
3/45
6.667
14/66
21.212
7/65
10.769
10/50
20
14.995±6.178
CD19
13/96
13.542
17/107
15.888
25/146
17.123
20/114
17.544
12/47
25.532
17.926±4.528
Supplementary Figures
Fig. S1 Karyotype of hEMSCPCs. Chromosome detection of hEMSCPCs between
passages 30 to 32 confirmed the karyotype as 46, XY.
Fig. S2 FISH of human Y chromosome in chimeric mice and immunofluorescent
images using tissue-specific antibodies. The human Y chromosomes appear as red spots as
revealed by FISH, while the FITC-labeled tissue-specific antibody appears green. In chimeric
mice, peripheral blood was positive for the human Y chromosome and human CD3 (A),
human CD16 (B) and human CD19 (C). Brain tissue from chimeric mice was positive for
both human Y chromosome and human MAP-2 (D) and human β-III tubulin (E). Cardiac
tissue from chimeric mice was positive for both human Y chromosome and human troponin I
(F). Lung tissue from chimeric mice was positive for both human Y chromosome and human
SP-C (G). Lung tissue from chimeric mice was positive for both human Y chromosome and
human CD31 (H). Splenic tissue from chimeric mice was positive for human Y chromosome
and human CD16 (I). Liver tissue from chimeric mice was positive for both human Y
chromosome and human ALB (J). Kidney tissue from chimeric mice was positive for both
human Y chromosome and human VEGF-R2 (K).