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THE SPINE TODAYInternational Congress
Rome Spine 6-7 dec 2011INTERVERTEBRAL
DISC REGENERATION
Helena Barreto Henriksson PhD,
researcherHelena Brisby assoc.
Prof,MD
Institute of Clinical Sciences\Orthopaedics
Gothenburg University/Sahlgrenska
University hospitalSweden
Regenerative medicine and tissue engineering is a rapidly increasing research field
Stem cells are involved in manyaspects of this field
Presentation outline
•Part II diagnostics, biological treatment options,from preclinic to clinical applications for cell therapy
•Part I disc anatomy, stem cells, stem cell niches, preclinical experiments, xenotransplantation
Helena Barreto Henriksson
Helena Brisby
•The intervertebral disc
Intervertebral disc-early development
4th week ,15 mm embryo
Image based on Peacock et. al J Anat , 1951, Christophersson et al. Am J Neuroradiol, 1999
5th week full term foetus
background
migration
Anatomy of the adult intervertebral disc
• support of our movements
• bearing/absortion of load
• fibrocartilage tissue
• chondrocyte like cells
• extracellular matrix
• nutrition/diffusion/endplates
Thesis, Henriksson 2010http://hdl.handle.net/2077/22925
background
Matrix components contribute to the flexibility of the intervertebral disc
chondrocyte like cell
collagen
proteoglycansglycosaminoglycans (GAGs)
hyaluronan
link protein
mobile ions
Thesis, Henriksson 2010http://hdl.handle.net/2077/22925
background
Characteristics of the degenerated disc
• loss of water content
• altered matrix composition
• reduced disc height
• loss of boundary beetwennucleus pulposus/annulus fibrosus
• dysfunctional cells
background
A) normal annulus fibrosus, female age 14 B) normal nucleus pulosus, female age 14C-D) degenerated disc, male age 44
• Stem cells/stem cell nichesStem cells are found in stem cell niches
References :Vogt.G, Hidden treasures in stem cells of indeterminately bilateral invertebrates, 2011Mitsiadis. T.A et al. Stem cell niches in mammals, 2007Poulsom et al. Adult stem cell plasticity, 2002Ohlstein, B. et al. The stem cell niche theme and variations, 2004
background
Where can stem cell niches be found?In mammals:
• bone marrow• skin• intestine• brain• and more…
What is a stemcell niche?
Ohlstein B, Spradling ACurrent opinion in cell biology, 2004
• An microenviromental structure that
nurtures and contains stem cells
• A specific localisation where stem cell
is resident, migrating
background
Common stem cell features
• ”self renewal”
• asymmetric cell division
• migration ability
• ”slow cycling” cells
• reacts to local and
distant signals
background
From stem cell to differentiated (mature) cells..
-asymmetric cell division-genome maintained vs daughter cells
-fast proliferating
-immature
-fully differentiated cells
-specialized functions
background
Differentiation capability of bone marrow mesenchymal stem cells
adult bone marrow multipotent stemcells (MSCs)
hemapoetic stem cell
non-hemapoetic stem cell
Thesis, Henriksson 2010http://hdl.handle.net/2077/22925
background
Normal regeneration of the intervertebral disc?
• Local stem-/progenitor cell populations in the disc?
Hypothesis- local stem cells support normal growth and regeneration of the disc
• local stem cells (STRO-1+, Notch1+ CD105, CD90)
• niches
• cellular migration
• rabbit model
background
Knee jointThornemo M et al. Journal of Anatomy, 2009Van Osch et al. Tissue Eng,2000Shapiro et al, J Bone Joint Surg Am, 1977Ranvier , Acad Sci Paris, 1873
A similar structure identifiedin the knee joint
niche
niche
Henriksson H et al. Spine, 2009
In vivo labeling of DNA- a method to study cell proliferation, label retaining cells and cell migration
• rabbit model, 2 age groups
• BrdU- peroral
• BrdU incorprated DNA- mitosis
-all tissues
• after labeling-the BrdU signal/cell gradually washed out-cell divisions
• labeled cells traced in different time points in tissues- antibody
ACT
T
G
GTA
CUAGC
GC TA
A
BrdU
BrdU= 5-bromo-2-deoxyuridine
microscopyevaluation
background
B
D
A D
NP
A2
A1EA1C
B1
C2
D1
D2A2
C1
P
4 days 10 days 10 days
NP
NP
AF
AF
AFo
AFo
P
P
AF
NP
AF
AFo
10 days 10 days
10 days 4 days rabbit4/18
13/1814/18
18/18
niche
28 days 56 days56 days 28 days
NPAF
AFoP
AFo niche
NP
Regions with stem cell niche like cell proliferation pattern found in outer annulus fibrosus and adjacent to the epihyseal plate
results
In vivo labeling with BrdU in rabbit intervetebral discs (n=18)
Local stem-/progenitor cellsfound in both non-degenerated and degenerated intervertebral discs
• Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region. A study in four species Henriksson et al. SPINE 2009
• Isolation and characterization of mesenchymal cells from human degenerated discs comparison with bone marrow mesenchymal stromal cells
Blanco et al. SPINE 2010
• Intervertebral discs derived stem cells: Implications for regenerative medicine and neural repair (canine)
Erwin et al. Abstract ISSLS 2011
STRO1 CD90
female, age 13 yearsfemale, age 61 years
CD105
male, age 41 years
results
• Cellular activities in the intervertebral disc?
Migration?
Hypothetical migration route-progenitor cellsBrdU+ cells counted in 4 different regions (16 regions/animal) at different time points
EMT=epithelial-mesenchymal transitions
BrdU labelling- group 1 age 3-5 months
rabbit, age 9 monthsrabbit, age 3 monthsAF AF
%
%
BrdU labelling - group 2 age 9-11 months
niche
nicheresults
MR= migration routeAFb=annulus fibrosus border region
EP= epihyseal plateAF= annulus fibrosus
mr1 mr2
AFb
mr1mr2
AFb
SNAI1 Isotype control total goat IgG
rabbit, age 3 months
nichearea
AFb
MR2MR1
rabbit 11 months
MR1
MR2
niche area
GDF5
Henriksson et al, SPINE,2011, IN PRESS
Migrating cells and prechondrocytic markers observed in normal intervertebral discs in the potential niche area
results
GDF5
• SNAI+, SLUG+ cellscytoskeleton migratory phenotype
• prechondrocytic markergrowth and differentiation factor 5 (GDF5)+ cells
• migration/cell activity normal rabbit, pig discs human degenerated discs
Outcome of analysis oflocal stem cells-/progenitor cells in the disc
• cells expressing classical mesenchymal stem cell markers detected in the intervertebral disc
• potential stem cell niches identified in the intervertebral disc region
• cellular activity-prechondrocytic markers, migration
Transplantation of stem cells into degenerated intervertebral discs
In vitro preclinical experiments
Isolation of bone marrow stem cells (MSCs)
• bone marrow sample aspirated from iliac crest
• 1/10 000- 1/100 000 - stem cell
• special centrifugation
• cell culture- adherence
• test of purity of cell
population (cell surface markers)
Positive effects on matrix formation?
Stem cell-/disc cell pellet in co-culture system with cartilage like apperance
human disc cells human stem cells
• monolayer cell cultures
• cell pellets co-cultured in chondrogenic media in a 3D cell culture system
Are mesenchymal stem cells and disc cells compatible cell types?
Increased matrix production was observed in the co-cultured cell pellets
disc cells MSCs
Proteoglycans (Gags) blue color (Alcian Blue Van Gieson staining)
Collagen II brown color (immunohistochemistry)
m25d75 m50d50 m75d25m25d75 m50d50m25d75
culture sets-one celltype co-culture sets 4/5
Patient 1
Patient2
Proteoglycans(blue)
Collagen II(reddishbrown)
Collagen II(reddishbrown)
Proteoglycans(blue)
_________________________________________________________
Co-culturesNucleuspulposus(np) and Mesenchymal(m) cells in 3D pellett-masssystem
mm25d75m50/np50m25/np75np
n=54/5
n=51/5
Results
MSCs 1/5
Henriksson et al, SPINE,2009
CollagenII
Gags
deg disc cells 25% stem cells 75%
deg disc cells 50% stem cells 50%
deg disc cells 75% stem cells 25%
7 days 14 days 28 days
Bars above the dotted line produced more GAG/DNA compared to degenerated disc pellets (one cell type) from donors respectively * = p<0.05 ** = p<0.01
Increased proteoglycan production observed in co–cultured pellets by biochemical analyses
Svanvik et al, Cell Tissues and Organs,2010Statistics: Anova and Mann Whitney U test
results
Outcome of in vitro experiments
• mesenchymal stem cells and disc- cells are compatible cell types
• increased matrix formation was obtained in the co-culture sets
Transplantation of stem cells to degenerated intervertebral discs
In vivo preclinical experiments
•injury/degeneration model (nucleus aspiration)
•after 2 weeks: transplantation (injection) of human stem cells (MSCs) 0.5 x 106 cells/porcine disc •carriers: hydrogel, cell culture medium
• no immuno-supprimental drugs
• experimental time period 6 months
In vivo experiments – xenotransplantation mini pig model
human mesenchymal stem cells (MSCs)
DMEM/F12 ,10% human serum, 10ng/mL,FGF
Henriksson et al, SPINE,2009
normal
6 months after stem cell transplantation
c/m norm c/g inj
3 months after stem cell transplantation
c/g injnormc/m
cells/ medium(c/m)
injured disc (inj)
cells/ hydrogel (c/g)
normal (norm)
Positive disc appearance on MRI after stem cell
transplantation with hydrogel carrier or cell medium
Henriksson et al, SPINE,2009
results
Positive control
A
6 months cell/gel 3 months cell/gel
E F H
Negative controlnormal pig disc
6 months, cell/med 3 months cell/medPositive controlhuman skin
B D
1 month cell/med
A
human cells (green)
• tracing performed with anti human antibodies (IHC)
• human cells detected-all animals after transplantation
• as solitary cells or incluster formations
human skin
normal pig disc
transplanted disc
transplanted disc
positive control
negative control
Human cells and matrix detected in transplanted discs – hydrogel or cell media
results
A B
Cell/gel 3 months
green= human cellsred= human collagen IIblue= nuclei (DAPI)
pig
pig
• detection of human collagen II at time points 3 and 6 months
• collagen II expressiondetected in transplanted discs
• differentiation of mesenchymal stem cells to chondrocyte-like cell type
negative control
human collagen IIanti human
Cell/gel 3 months
pig
pig
Human cells and matrix detected in transplanted discs –hydrogel or cell media
results
normal disc
cell/media transplanted disc cell/gel transplanted discCOL L AG EN I
1
10
100
1000
10000
nega
tivco
ntrol
cell/m
ed
cell/g
el
nega
tivco
ntrol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
relat
ive v
alue
AGGRECAN
0,1
1,0
10,0
100,0
1000,0
10000,0
nega
tivco
ntrol
cell/m
ed
cell/g
elne
gativ
contr
olce
ll/med
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive
valu
e
VERSICAN
1
10
100
1000
10000
nega
tivco
ntrol
cell/m
ed
cell/g
el
nega
tivco
ntrol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive
value
COLLAGEN 2A
1,E+00
1,E+04
1,E+08
1,E+12
nega
tivco
ntrol
cell/m
ed
cell/g
elne
gativ
contr
olce
ll/med
cell/
med
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive
valu
e
COLLAGEN 2B
1,E+00
1,E+04
1,E+08
1,E+12
nega
tivco
ntrol
cell/m
ed
cell/g
elne
gativ
cont
rol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive
valu
e
SOX9
110
100
100010000
100000
nega
tivco
ntrol
cell/m
ed
cell/g
el
nega
tivco
ntrol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive
valu
e
COLLAGEN I
1
1 0
1 001 0 0 0
1 0 0 0 0
nega
tivco
ntro
l
cell/
med
cell/
gel
nega
tivco
ntro
lce
ll/m
ed
cell/
med
cell/
gel
cell/
gel
cell/
gel
3 mo n th s 6 mo n t h s
relat
ive va
lue
A G GR EC A N
0 , 1
1 , 0
1 0 ,0
1 0 0 , 0
1 0 0 0 ,0
1 0 0 0 0 ,0
nega
tivco
ntro
lce
ll/m
ed
cell/g
el
nega
tivco
ntro
lce
ll/med
cell/
med
cell/
gel
cell/
gel
cell/
gel
3 m o n th s 6 mo n th s
rela
tive v
alue
V ERSIC A N
1
1 0
1 0 01 0 0 0
1 00 0 0
nega
tivco
ntro
l
cell/
med
cell/g
el
nega
tivco
ntro
l
cell/
med
cell/
med
cell/g
el
cell/
gel
cell/
gel
3 mo n t h s 6 m o n th s
rela
tive v
alue
CO L LA GE N 2A
1, E+0 0
1, E+0 4
1, E+0 8
1, E+1 2
nega
tivco
ntro
lce
ll/m
ed
cell/
gel
neg
ativ
cont
rol
cell/m
ed
cell/
med
cell/
gel
cell/
gel
cell/
gel
3 mo n th s 6 m on t h s
rela
tive
valu
e
CO L LA G EN 2 B
1 ,E+ 0 0
1 ,E+ 0 4
1 ,E+ 0 8
1 ,E+ 1 2
nega
tivco
ntro
l
cell/
med
cell/
gel
nega
tivco
ntro
l
cell/
med
cell/
med
cell/g
el
cell/g
el
cell/g
el
3 mo n th s 6 mo n th s
rela
tive
valu
e
SOX9
11 0
10 01 0 0 0
1 0 0 0 01 0 0 0 00
nega
tivco
ntro
l
cell/
med
cell/
gel
nega
tivco
ntro
lce
ll/m
ed
cell/
med
cell/
gel
cell/
gel
cell/
gel
3 mo n th s 6 mo n t h s
relat
ive v
alue
COLLAGEN I
110
1001000
10000
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 months
re
lati
ve
va
lue
AGGRECAN
0,1
1,0
10,0
100,0
1000,0
10000,0
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 months
re
lati
ve
va
lue
VERSICAN
110
1001000
10000
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 months
re
lati
ve
va
lue
COLLAGEN 2A
1,E+00
1,E+04
1,E+08
1,E+12
neg
ati
v
co
ntr
ol
ce
ll/m
ed
cell
/ge
l
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
cell
/med
ce
ll/g
el
cell
/ge
l
ce
ll/g
el
3 months 6 months
rela
tiv
e v
alu
e
COLLAGEN 2B
1,E+00
1,E+04
1,E+08
1,E+12
neg
ati
v
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
neg
ati
v
co
ntr
ol
cell
/med
ce
ll/m
ed
ce
ll/g
el
cell
/ge
l
cell
/gel
3 months 6 months
re
lati
ve
va
lue
SOX9
110
1001000
10000100000
neg
ati
v
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
cell
/med
ce
ll/g
el
cell/g
el
ce
ll/g
el
3 months 6 months
rela
tiv
e v
alu
e
COLLAGEN I
1
10
1001000
10000
ne
ga
tiv
co
ntr
ol
cell/m
ed
cell/g
el
ne
ga
tiv
co
ntr
ol
cell/m
ed
ce
ll/m
ed
cell/g
el
cell/g
el
ce
ll/g
el
3 months 6 months
rela
tive v
alu
e
AGGRECAN
0,1
1,0
10,0
100,0
1000,0
10000,0
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 monthsre
lati
ve v
alu
e
VERSICAN
110
1001000
10000
ne
gati
v
co
ntr
ol
cell/m
ed
cell/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive v
alu
e
COLLAGEN 2A
1,E+00
1,E+04
1,E+08
1,E+12
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 months
rela
tive
va
lue
COLLAGEN 2B
1,E+00
1,E+04
1,E+08
1,E+12
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 months
rela
tiv
e v
alu
e
SOX9
110
1001000
10000100000
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
ne
ga
tiv
co
ntr
ol
ce
ll/m
ed
ce
ll/m
ed
ce
ll/g
el
ce
ll/g
el
ce
ll/g
el
3 months 6 months
rela
tiv
e v
alu
e
COLLAGEN I
1
10100
100010000
ne
gati
v
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
neg
ati
v
co
ntr
ol
ce
ll/m
ed
cell/m
ed
ce
ll/g
el
cell/g
el
ce
ll/g
el
3 months 6 months
rela
tiv
e v
alu
e
AGGRECAN
0,1
1,0
10,0
100,0
1000,0
10000,0
neg
ati
v
co
ntr
ol
cell/m
ed
cell/g
el
neg
ati
v
co
ntr
ol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive v
alu
e
VERSICAN
110
1001000
10000
neg
ati
v
co
ntr
ol
ce
ll/m
ed
ce
ll/g
el
neg
ati
v
co
ntr
ol
ce
ll/m
ed
cell/m
ed
ce
ll/g
el
ce
ll/g
el
cell/g
el
3 months 6 months
rela
tiv
e v
alu
e
COLLAGEN 2A
1,E+00
1,E+04
1,E+08
1,E+12
neg
ati
v
co
ntr
ol
cell/m
ed
cell/g
el
neg
ati
v
co
ntr
ol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive v
alu
e
COLLAGEN 2B
1,E+00
1,E+04
1,E+08
1,E+12
negati
v
co
ntr
ol
cell/m
ed
cell/g
el
negati
v
co
ntr
ol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive v
alu
e
SOX9
110
1001000
10000100000
negati
v
co
ntr
ol
cell/m
ed
cell/g
el
neg
ativ
con
trol
cell/m
ed
cell/m
ed
cell/g
el
cell/g
el
cell/g
el
3 months 6 months
rela
tive v
alu
e
COLLAGEN IIB
Human gene expression of matrix markers in porcine discs 3 and 6 months after stem cell transplantation
• human cartilage gene markers collagen IIA and IIB
aggrecan, versican
sox9
collagen I
• viable cellsRNA short degrading time
• functional cellsstem cells differentiated into chondrocyte-like cells
matrix accumulation
results
Henriksson et al, SPINE,2009
VERSICAN
AGGRECAN
Outcome of in vivo studies
• the transplanted stem cells were viable (6 months) and functional
• differentiation toward a chondrocyte–like cell type
• matrix formation obtained
• positive MRI findings
• hydrogel carrier provide an attachment network for cells
Ref: Wang et al. Journal of Orthopaedic Science, 2010
Outcomes of stem cell transplantation into injured/degenerated discs in different animal models
Thank you for listening!
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