New Insights into Paracrine Activity of Stem Cells
in Heart Repair
- Microvesicles and Proteomics
Ewa K. Zuba-Surma, PhD, DSc
Department of Cell Biology
Faculty of Biochemistry, Biophysics and Biotechnology
Jagiellonian University, Krakow, Poland
Infarcted Heart – Target for stem cell therapy
Stem cell populations for heart repair
Tongers et al. Eur Heart J (2011)
BM-derived SC - in vivo regeneration
Sca-1+Lin-CD45-
GFP+
(VSELs)
LV Ejection Fraction
* P < 0.05 vs. Vehicle-treated
†
P < 0.05 vs. HSCs
%
*†
*†
*†
Gr I (Control/ Vehicle)
Gr II (HSCs)
Gr III (VSELs)
BSL
48 h
1 mo
3 mo
6 mo
Dawn B et al. Stem Cells 2008
Zuba-Surma EK et al. J Mol Cell Cardiol 2010
Paracrine effects of transplanted VSELs
VSELs
Differentiating to CMs
Microarrays (mRNA)
Wojakowski et al. Int J Oncol. 2010;37(2):237-47
Zuba-Surma et al. Antioxid Redox Signal. 2011 May 5.
Goals for stem cell therapy in heart repair
Microvesicles (MVs)
“new players on the board”
Ratajczak et al. Leukemia 2011, Dec 19.
Gnecchi M. Circ Res. 2008; 103(11): 1204–19
Burchfield JS & Dimmeler S. Fibrogenesis & Tissue Repair 2008,1:4.
Microvesicles (MVs) – carriers of biactive content
ü Membrane receptors, adhesions, lipids, enzymes
ü Cytoplasmic proteins (e.g. enzymes, TFs)
ü Nucleic acid (DNA, mRNAs, miRNAs)
Muralidharan-Chari et al. J Cell Sci 2010, 123:1603-11.
Stem cells as source of MVs
iPS-MVs
MVs
?
Corsten et al. 2008 Lancet Oncol.
http://www.eurostemcell.org
Aim of study:
May iPS- derved MVs play a role in cell-to-cell transport and
affect target cell function?
May they play a potential role in heart regeneration via
paracrine activity?
„Serum- free/ Feeder-free” hiPS cells
hiPS reprogramming and culture
Seeding
UC-MSC
Transduction
Sendai virus (OSKM)
0
1
3
5
7
9
12
17
Hypoxia (5% O2) + inhibitors
30
hiPS phenotype (PSC markers) by IHC, FC
Tra-1-60
Control
hiPS
97,8%
FSC
FSC
0,1%
SSEA-4
25
Further characterisation of iPS clones
(TGFβi; GSK3βi; MEKi; ROCKi)
PALP
21
OCT4-PerCP-Cy5.5
OCT4-PerCP-Cy5.5
NANOG-PE
-1
Picking iPS colonies
for expansion
Medium change to Essential 8;
growing cells on matrigel
hiPS
99,8%
SOX2-APC
hiPS – multiantigenic phenotype by FC
SSEA-1
CD45
0.3%
hiPS – karyotype by Affimetrix
CD106
26%
hiPS
0.5%
11
1
CD133
CD81
99%
2
3
4
5
6
7
8
9
10
13 14
15
16
17
18
19
20
21
22
12
KDR
99%
77%
X
Y
UC-MSCs
11
CD90
CD29
CD324
99%
99%
99%
1
2
3
4
5
6
7
8
9
10
13 14
15
16
17
18
19
20
21
22
12
X
Y
hiPS – derived MVs
MV Isolation - Sequential ultracentrifugation
hiPS
culture
Condition media
collection
MVs
pellet
2,000xg
(15min)
100,000xg
(60 min)
Centifugation
Ultracentifugation
(2x)
MV Evaluation - Nanoparticle Tracking Analysis (NTA)
hiPS-MVs
UC-MSCs
hiPS- derived mRNAs and miRNAs are present in hiPS-MVs
Selected miRNAs
Selected mRNAs
Exogenous transcripts are exported to hiPS-MVs
hiPS cells overexpressing GFP and miR-1
B. hiPS-EGFP
A.
LV vector
C.
hiPS: WT
hiPS-miR-1-copGFP
GFP miR-1 Ctrl
M a b a b a
b C+ C-
EGFP
copGFP
a.
b.
CC+
M
hiPS cells
hiPS-MVs
Water control
GFP plasmid control
DNA ladder
Exogenous transcripts are exported to hiPS-MVs
miR-1 overexpression in hiPS cells and hiPS-MVs
A. miR-1
Sylwia Bobis-Wozowicz
13.12.2013Krakow
B. miR-16
(control)
CT ratio
CT ratio
CT in hiPS-MVs /
CT in hiPS cells
CT in hiPS-MVs /
CT in hiPS cells
hiPS-MVs are transferred to target cells (primary hcMSCs)
MV transfer (EGFP-hiPS-MVs to primary human cardiac MSCs)
hcMSC
(Uptaken MVs)
hcMSC
culture
+ GFP- MVs
(50ug)
1h incubation
+ DiI dye
(0.1ug/ml)
3min incubation
Internalized MVs (GFP+/DiI-)
Extracellular MVs (GFP+/DiI+)
Endogenous mRNAs are transffered to traget cells via hiPS-MVs
Sylwia Bobis-Wozowicz
13.12.2013Krakow
mRNA transfer assessment (hcMSCs + 50ug of hiPS-MV)
∆∆CT
Selected mRNAs in hcMSCs by qRT-PCR
hiPS-MV treatment may enhance hcMSC proliferation
MTT Proliferation assay (hcMSCs + 50ug of hiPS-MV)
hcMSCs proliferation following incubation with hiPS-MVs by spectroscopy
**
**
OD [Absorbance]
**
*
Control
MVs+/MVs+
N
H
cMSC untrated with MVs
cMSC treated with hiPS-MVs for 22h (washed twice with PBS)
cMSC treated with hiPS-MVs for entire experiment
normoxia
hypoxia
*p<0.01
**p<0.001
Summary and Conclusions
Sylwia Bobis-Wozowicz
13.12.2013Krakow
ØhiPS-MVs are rich in various miRNAs and mRNAs
Ø Endogenous mRNAs from hiPS cells can be transferred to target cells
via hiPS-MVs
Ø Exogenous RNAs (mRNA/miRNA) are exported from hiPS cells to MVs
ØhiPS-MVs may affect function of target cells including proliferation (under
evaluation)
1. MVs may represent a vast arm of SC paracrine activity and potentially affect
functions of endogenous heart cells following transplantation
1st Poster Award
Acknowledgments
„MicroTEAM”
Department of Cell Biology, Faculty of
Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Krakow, Poland
dr hab. Ewa Zuba-Surma
dr Sylwia Bobis-Wozowicz
mgr Marta Adamiak
mgr Elżbieta Kamycka
mgr Anna Łabędź-Masłowska
Agnieszka Pająk
Katarzyna Kmiotek
FNP Team Project
„Biactive stem cell- derived microvesicles as novel tool for tissue regeneration”
POIG: „Innovative methods for stem cell applications in medicine”
Acknowledgments
Department of Cell Biology
Faculty of Biochemistry, Biophysics
and Biotechnology,
Jagiellonian University, Krakow, Poland
Cardiovascular Research Institute,
Kansas University, Kansas City, KA, USA
Zbigniew Madeja
Stem Cell Institute,
University of Louisville, KY, USA
Mariusz Z. Ratajczak
Magda Kucia
III Clinic of Cardiology
Silesian Medical University
Katowice, Poland
Buddhadeb Dawn
Michał Tendera
Wojtek Wojakowski
Institute of Molecular Cardiology,
University of Louisville, KY, USA
Department of Pediatric Cardiac Surgery,
Polish-American Children’s Hospital,
Jagiellonian University, Krakow, Poland
Roberto Bolli
Jacek Kolcz
Laboratory of Cell and Gene Therapy
Department of Transfusion Medicine
University Medical Centre Freiburg, Germany
Polish Stem Cell Bank S.A.
Tony Cathomen
Jagiellonian Centre for Experimental Therapeutics
(JCET)
BM- derived adult Very Small Embryonic-Like SC (VSELs)
Sca-1+Lin-CD45Ø Non-hematopoietic SC in BM
Ø Very small- sized cells with expression of embryonic antigens
(e.g. Oct-4, Nanog, Rex-1, SSEA-1/4)
Ø Low immunogenity (optimal for allotransplantations)
Ø Possess wide differentiation potential (Multi-/Pluripotent SCs)
Oct-4 promotor methylation
EB- like colonies
1μm
SEM
PALP
Ectoderm
(pancreatic cells)
TEM
Endoderm
(neural cells)
Mesoderm
(cardiomyocytes)
Shin DM et al. Leukemia 2009
Zuba-Surma EK et al. J Cell Mol Med 2008
VSELs: Sca-1+/CD184+/CD34+/
Oct-4+/Nanog+/SSEA-1/4+/Tra-160/81+/CD106+-/Flk-1+-/CD45Kucia M et al. Leukemia 2006;20:857-869.