Innovative Stem Cell-based Approaches for
Bone and Skin Tissue Engineering
Alexandra P. Marques
1
3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho,
Headquarters of the European Institute of Excellence on Tissue Engineering and
Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal
2
IBB – Institute for Biotechnology and Bioengineering, PT Associated Laboratory, Guimarães,
Portugal
Tissue Engineering
II. Expansão das células
I. Obtenção de células
estaminais do próprio paciente
(da medula óssea ou do tecido
adiposo, por exemplo)
in vitro
III. Desenvolvimento da estrutura
de suporte (biomaterial polimérico)
para a criação de um substituto de
tecido
V. Implantação do substituto do
tecido no defeito do paciente
IV. Cultura das células no material de
suporte
in vitro até se obter um
substituto de tecido-material híbrido
funcional
Levels of Cellular Communication
Soluble Factors
Cell-Cell Contact
Cell-ECM Interactions
Cell Sheet Engineering
Poly (N-isopropylacrylamide) grafted culture dishes (T. Okano)
adapted from M. Hirose et al. Yonsei Med J 41 (2000) &
J. Yang et al. Biomaterials 26 (2005) 6415–6422
Cell Sheet Engineering for Bone TE
Homogenization
In Vitro Characterization
•H&E
•Osteocalcin Expression
•Mineralization (Alizarin
Red)
Bone Marrow aspirates
Mononuclear
Fraction
Differential
Centrifugation
In Vivo Implantation
Cell Sheets
(after 3 weeks in
osteogenic medium)
Selection by TCPS
adhesion
Bone Marrow
Mesenchymal Precursor
Cells (BM MPC)
•Subcutaneously in the
dorsal flap of nude mice
•For 7 days, 3 and 6 weeks
In Vivo Characterization
Seeding on
Thermoresponsive Discs
Expansion
•H&E
•Osteocalcin Expression
•Mineralization (Alizarin
Red)
RP PIrraco, AP Marques, RL Reis, M Yamato, T Okano, submitted
Cell Sheet Engineering – In Vitro Analysis
Osteocalcin
H&E
Mineralization
RP PIrraco, AP Marques, RL Reis, M Yamato, T Okano, submitted
Cell Sheet Engineering –In Vivo Osteocalcin Analysis
7d
3W
6W
6W Control
RP PIrraco, AP Marques, RL Reis, M Yamato, T Okano, submitted
Cell Sheet Engineering –In Vivo MineralizationAnalysis
7d
3W
6W
6W Control
RP PIrraco, AP Marques, RL Reis, M Yamato, T Okano, submitted
Skin
 Epidermis:
melanocytes.
Keratinocytes
and
 Dermis: Fibroblasts, endothelial cells
(which will form the vascular system of the
dermis).
http://www.fpnotebook.com/DER/Exam/SknAntmy.htm
http://journals.prous.com/journals/servlet/xmlxsl/pk_journals.xml
Current Strategies to Engineer Skin
Epidermis Analogues
Dermis Analogues
Epicell
Laserskin
Integra
Combined Products
Full-Thickness
Wounds???
Apligraf
http://www.fda.gov/cdrh/mda/docs/H990002.html
http://ec.europa.eu/research/press/2000/pr2703en-an.html
http://www.devicelink.com/mddi/archive/98/05/018.html
http://www.businessfacilities.com/blog/labels/Site%20Selection.html
Stem Cells in Skin TE: Revolutionary Approach
Self-renewal capacity
Multi-lineage Differentiation
Unlimited Biological
Material
Large Skin
Defects
http://stemcells.nih.gov/
Epidermal
Adult Stem Cells
http://www.promocell.com
Embryonic Stem Cells
Endothelial
http://www.invitrogen.com
Well defined and efficient protocols for directing stem cell differentiation
Main skin cell lineages
Human Amniotic Fluid Stem Cells (hAFSCs)
Amniotic Fluid from Amniocentesis
Genetics
Department of the
Faculty of Medicine
of the University of
Porto
Culture supernatants (day 6)
Culture of hAFSC to confluency
Nat Biotechnol. 2007 Jan;25(1):100-6
Plating at 500 and 1000 cells/cm2
hAFSCs Phenotype (P5  P16)
Positive
Negative
CD29
CD44
CD34
CD73
CD90
CD105
CD45
AM Frias…..RL Reis, Tissue Engineering A (2008) 387
Human Adipose Stem Cells (hASCs)
Human Adipose Stem Cells (hASCs) Isolation
 Lipoaspirate with Collagenase II
 Centrifugation for 10 minutes at 1200 rpm.
 Erythrocyte lysis buffer
 Pellet ressuspension DMEM (10% FBS,
1% Antibiotic).
CD29
Hospital da Prelada, Porto
CD105 / CD73
Primary Skin Cells Isolation
Human skin samples were
obtained from abdominoplasty
procedures
of
healthy
volunteers, within the compass
of the protocol with the Prelada
Hospital at Porto.
Human Keratinocytes
100 m
100 m
Human dermal fibroblasts
Differentiation Strategies
Stem Cells
hASCs & hAFSs
Indirect CoCulture
(Transwells®)
Direct
Co-culture
(DC)
Keratinocytes
Conditioned
Medium (CM)
Differentiated Cells?
Exogenous
Cytokine
Cocktail
Stem and Keratinocyte Markers
hAFSCs
hKc
CD90
p63
cytokeratin 1/10
CD 29
DAPI
DAPI
DAPI
cytokeratin 19
cytokeratin 14
DAPI
DAPI
CD 106
CD 44
DAPI
(+) p63, cytokeratin 19, cytokeratin 14 and cytokeratin 1/10.
hASCs
CD90
(+) CD90, CD29, CD106, CD 44.
(-) CD 34, CD 45
CD105
CD73
hAFSCs Differentiation (CM) - Cell Morphology
50% CM
50% CM
30% CM
30% CM
7d
14d
7d
14d
100 m
100 m
100 m
100 m
50% CM with FBS
50% CM
30% CM
30% CM
7d
with FBS
with FBS
with FBS
14d
7d
14d
100 m
100 m
100 m
100 m
Culture in α-MEM
Culture in α-MEM
7d
14 d
Controls
100 m
100 m
[dsDNA] / (µg/ml)
hAFSCs Differentiation (CM) - Cell Proliferation
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
hAFSCs Differentiation (CM) – Cell Phenotype
50% CM no FBS
30% CM no FBS
50% CM with FBS
30% CM with FBS
Phalloidin
Phalloidin
Phalloidin
Phalloidin
DAPI
DAPI
DAPI
DAPI
p63
p63
p63
p63
Phalloidin
Phalloidin
Phalloidin
Phalloidin
DAPI
DAPI
DAPI
DAPI
Cytokeratin 1/10
Cytokeratin 1/10
Cytokeratin 1/10
Cytokeratin 1/10
Phalloidin
Phalloidin
Phalloidin
DAPI
DAPI
DAPI
DAPI
Cytokeratin 19
Cytokeratin 19
Cytokeratin 19
Cytokeratin 19
Phalloidin
Phalloidin
Phalloidin
Phalloidin
DAPI
DAPI
DAPI
DAPI
Cytokeratin 14
Cytokeratin 14
Cytokeratin 14
Cytokeratin 1/10
Phalloidin
hASCs Differentiation (CM) – Cell Proliferation
75%
100%
50%
75%
100%
14 days
7 days
50%
Diminished Proliferation
Increasing proliferation along the time of culture was
observed in the Transwells® system and in the
composed culture medium.
hASCs Differentiation - Cell Phenotype
7 days
p63
hASCs with 100% KC-CM 48h
Exogenous expression of p63 at day 7 and 14.
Expression of CKRT 14 at day 14, but not at day 7.
Low Proliferation
CKRT14
14 days
p63
hASCs in DMEM +
10ng/mL KGF + 25ng/mL
EGF + 60 ng/mL IGFII
Direct culture of hASCs and hKc
Ratios (hASCs: hKc)
1:1
1:10
R1
-KSFM
-KSFM/DMEM
-KSFM/DMEM+FBS
99.02%
M2
Dil Labeling
1:1 KSFM
1:10KSFM
1:10 KSFM/DMEM
1:10 KSFM/DMEM/ FBS
hASCs Differentiation (DC) - Cell Phenotype
Cytokeratin 14 expression in 1:1 KSFM
Differentiated hASC
hASCs
keratinocyte
4.09%
10d
10d
hASCs Differentiation (DC) - Cell Phenotype
Cytokeratin 14 expression in 1:1
KSFM/DMEM
10d
0.76%
0.87%
Cortar!
hASCs Differentiation (DC) - Cell Phenotype
Cytokeratin 14 expression in 1:1
KSFM/DMEM/FBS
hASCs Differentiation (DC) - Cell Phenotype
%
CKTR14+Dil-
1:1
1:10
5d
10d
5d
10d
KDFBS
KSFM
KD
ND
0.89
0.76
ND
4.09
0.87
ND
ND
ND
ND
ND
ND
CKRT14
DiI
DAPI
Conclusions
Rat bone marrow MSCs-derived cell sheets differentiated in PIPPAm
culture dishes promote the formation of new bone tissue after in vivo
implantation; New bone tissue formed in vivo presents a marrow
morphologically similar to bone marrow
Indirect Co-culture systems, using Transwells®, are not sufficient to trigger
early Epidermal Differentiation of hASCs cultures while in KSFM, direct
contact of KC and hASCs induce the expression of early epidermal
markers by stem cells.
hKc conditioned medium induces the expression of early epidermal
markers by hAFSCs.
 These herein presented strategies have demonstrated potential for further
bone and skin tissue engioneering studies.
Aknowledgements
European Union NoE
EXPERTISSUES
(NMP3-CT-2004-500283)
www.expertissues.org
__________________________________
Cooperation agreement with Hospital de S. Marcos, Braga, and
Hospital da Prelada and Faculty of Medicine, Porto