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Adult stem cells
Rob Vries, Clevers group, Hubrecht Institute, 2012
Epithelial self-renewal in
the small intestine
Charles Philippe Leblond, (1910 – 2007)
Stem cell pioneer
1974: CBC (Crypt base columnar cells) are
stem cells
Lgr5-Driven
GFP
in Crypt Base
Columnar Cells
Multicolor lineage tracing
Rainbow (J.Lichtman) targeted into Rosa26 locus
Snippert et al., Cell 2010
Conclusion
Lgr5 stem cells are initial targets of oncogenic transformation
Stem Cell cultures
Sorted Lgr5-driven GFP+ cells from intestine
Lgr5-GFP
Single CBC cells form crypt/villus
structures in matrigel culture
Het zuiveren en
kweken van
lichtgevende
stamcellen.
Structures contain all
intestinal cell types
Mucin2
Villin
Lysozyme
Lumen
ChromograninA
Lumen
Lumen
Enterocytes
Goblet Cell
Paneth
EnteroEndocrine
Applications of Lgr stem cells
1) Stem Cell Transplantation
Small Intestine, Colon, Liver, Pancreas
2) Patient specific therapy prognosis
Tissues: Small Intestine, Colon, Liver, Pancreas,
Prostate, Lung, (starting with breast)
3) Drug screening
eg: CF,Cancer
Stem Cell transplantation
Long-term survival of
transplanted colon
organoids in vivo (with
Tetsuya Nakamura, Shiro
Yui and Mamoru Watanabe)
Transplanted region looks entire normal
Yui S et all.,
Nat. Med., 2012
Liver Stem Cells
Liver Organoids
Liver Stem Cell transplantation
(10x)
(20x)
Applications of Lgr stem cells
1) Stem Cell Transplantation
Small Intestine, Colon, Liver, Pancreas
2) Patient specific therapy prognosis
Tissues: Small Intestine, Colon, Liver, Pancreas,
Prostate, Lung, (starting with breast)
3) Drug screening
eg: CF,Cancer
Stem Cells for drugscreening
Prostate organoids
CK8
p63
AR
ENR
ENR + DHT
• Basal cells form outer
layer
• Luminal cells form inner
layer
• AR is expressed in
majority of cells
• Addition of DHT induces
polarization &
differentiation
Human Tissues
Prostate Organoids
Colon Cancer Organoids
Tumor
Normal
Cystic Fibrosis Cells for drugscreening
Healthy Control
Cystic Fibrosis Cells for drugscreening
Human small intestinal organoids
Cystic Fibrosis Cells for drugscreening
Cystic fibrosis patient
Induction of CFTR receptor by cAMP
Chemical compounds
Cystic Fibrosis Cells for drugscreening
restore CFTR-F508del
Cystic fibrosis patient + drugs
function
Chemical compounds
Correction of phenotype by CF drugs
restore CFTR-F508del
function
Dekkers et al., unpublished
Toshi Sato
Nick Barker
Johan van Es
Marc van de Wetering
Shiro Yui, Mamoru Watanabe (Tokyo)
Hugo Snippert
Apollo Pronk, Joost van Gorp, Winan van Houdt (Diakonesse
Laurens van der Flier
Hospital, Netherlands)
Meritxell Huch Ortega
Craig Dorell, Markus Grompe (Oregon)
Arnout Schepers
Markus Loeffler (Germany)
Wim de Lau
Ben Simons (Cambridge, UK)
Florijn Dekkers, Jeffrey Beekman, Kors van der Ent
UMC Utrecht, Netherlands
Hans Clevers
Single mRNA molecule in situ for putatuve crypt
stem cell markers (van Oudenaarden c.s., Nat Cell
Biol 2011): Bmi1 is broadly expressed.
Bmi1
Lgr5
Lgr5
Drug/ Target screening and validation
Training
IP
UMC Utrecht Living Biobank
Develops and/or license organoid
(screening) technology
Commercial Drug screening
1)
Creates a ‘Living’ Biobank of patient specific organoids
2)
Disease specific organoids with sequence, patient data, etc
3)
‘Paired’ organoids
patient sample of both normal and cancer
Academic research
Lgr5 stem cells arise upon tissue damage.
liver
Wnt signaling activation after injury
Wnt signaling activation after injury
Lgr5 cells express Sox9 ductal & progenitor
marker Lgr5 cells express Sox9 ductal &
progenitor marker
Single Lr5 positive (lacZ) cells grow into
organoids
Human stomach organoids
H&E
MUC6
MUC5A
C
PAS
Tissue
Organoid
s
-/- mouse injected
Liver cell
transplantation
into
FAH
withtyrosinemia
GFP1 clone.
Sacrificed
53 days
post-transplant
mouse model
(FAH-/model)
(with Craig Dorell, Markus Grompe)
0/40 mice show
ana/dysplasia
FAH-/- mouse injected with GFP1 clone.
Sacrificed 53 days post-transplant
(with Craig Dorell, Markus Grompe)
Growing adult
tissues
from
Liver
project
stem cells
Duncan GASTROENTEROLOGY 2009;137:466–481