Learning about human iPSC models for
glycosylation-related disease.
Stephen Dalton, Ph.D.
Department of Biochemistry and Molecular Biology
Paul D. Coverdell Center for Biomedical and Health Sciences
Stem cells in early development give rise to all the adult tissues
(pluripotent stem cells)
Pluripotent cells differentiate and become more specialized
stem cell
2 poten(al sources of pluripotent stem cells for research differentiation
cell specialization
IVF embryo
Embryonic stem cells (ESCs)
Donor cells
Induced pluripotent stem cells
(iPSCs)
specialized cell types
- neurons, muscle, liver, blood, pancreas cells etc.
Early cell fate specification of cultured pluripotent cells
Pax6
Sox1
Sox2
nervous system*
skin
Ectoderm
hair
neural crest*
hESCs/
hiPSC
Oct4
Nanog
Sox2
E-cad
specification
factors
MesEnd
Meso
PDGFRα
FoxF1
Nkx2.5
Isl1
T
Wnt3
Snail
MixL1
DE
Sox17, CXCR4
GATA4,6
blood
bone
muscle*
kidney
cardiovascular*
liver*
lung
pancreas*
stomach
thymus
thyroid
Diverse cell populations: tools for understanding CDG and CMD
4 A cell therapy pathway
IVF
embryo
ESC/iPSC
pluripotent
stem cell
Master cell bank
propagate
cells
Cell donor
Transplantable
cell
differentiate
cells
implant into
patients
Reprogramming: taking a specialized cell (from a patient)
and converting into a pluripotent stem cell
stem cell
de-differentiation
reprogramming
specialized cell types from patient/donor
- neurons, muscle, blood cells etc.
Shinya Yamanaka
Kyoto University
2012 Nobel Prize for Physiology or Medicine
Modeling CDG and CMD: the strategy of reprogramming
CDG or CMD patient iPSCs
CDG or CMD patient cells
Human iPSCs
A pathway to modeling human disease with iPSCs
source patient cells
amplify
reprogram
quality control: genotype
assess differentiation potential
confirm disease phenotype
potential drug screens
molecular analysis
functional analysis
(animal models)
Engineering mutations into iPSCs to create human
CDG and CMD models
•  single or multiple mutations can be introduced into
non-patient (normal) cells to model human disease
•  patient cells can be genetically corrected and used
as research tools
•  utility in drug screening (new therapeutics)
•  new tools to understand the molecular basis of
glycosylation disorders
CRISPR-Cas9 Genome Editing: 1. Gene Disruption
Guide RNA design Guide RNA plasmids 20 base guide RNA PAM U6 20 bp gRNA Scaffold gRNA 1 gRNA 2 ATG PST/STX/NCAM Genomic locus 300 bp 1kb CAG BFP Zeo IRES Cas9 Poly A Homology directed repair gRNA 2 gRNA 1 CAG 1kb BFP Zeo IRES Poly A Inser(on and gene disrup(on Terminator 2. Introduction or correction of mutations
Guide RNA design PAM 20 base guide RNA sgRNA ST3Gal5 exon 7 S&P ST3Gal5 genomic A (994) 60 bp LoxP CAG 1kb LoxP BFP IRES Cas9 Repaired ST3Gal5 gene Poly A Homology directed repair LoxP LoxP CAG G (994) Zeo BFP Zeo IRES ST3Gal5 exon 7 G (994) Poly A CRE Repaired ST3Gal5 gene LoxP ST3Gal5 exon 7 G (994) 1kb Modeling human disease with patient-derived induced pluripotent
stem cells (iPSCs)
Cells carrying a mutation(s)
responsible for congenital
disorder of glycosylation
iPSC
pluripotent
reprogram
cell donor with CDG
- skin biopsy
- blood
propagate
cells
Characterize glycosylation
defect at molecular level,
use for drug screening etc.
A pa(ent-­‐specific model for human disease differentiate
cells
Using iPSCs to understand congenital disorders of
glycosylation (CDG) and congenital muscular
dystrophy (CMD)
Mike Tiemeyer
Lance Wells
Rich Steet
Glycoconjugates on the cell surface of animal cells
“Salt and Pepper Syndrome” (S&P): applying glycomic technology
to investigating human disease mechanism
• identified in 1 African-American family in
the southeastern US at the Greenwood
Genetic Center, Greenwood, SC
• a missense mutation (p.E332K) was
identified in the ST3GAL5 (GM3 synthase)
gene of two siblings.
• profound intellectual disability
• failure to thrive
• seizure disorder
• mid-face hypoplasia
• scattered dermal hyper- and hypopigmentation
Charles Schwartz, Greenwood Genetic Center
Zebrafish
iPS cells
LL
O
Fibroblast
FOS
LL
NC cells
FOS
LL
O
ST3Gal5
FOS
O
LL
FOS
LL
O
Normal
O
Alteration of glycomic diversity in S&P syndrome
S&PS
Fibroblast
S&PS
Zebrafish
S&PS
iPS cells
S&PS
NC cells
GM3
absent
reduced
absent
absent
LacCer
-
↗
-
↗
Glycoprotein
sialylation
↗
↗
↗
-
High mannose
glycans
-
-
-
↗↗↗
Apoptosis
-
☹
-
☹
Generation of patient-derived hiPSCs
Oct4, Sox2, Klf4, c-­‐myc Sendai virus reprogram
hiPSC differentiated cells
(glycan analysis)
patient cells
Reprogrammed cells from ‘salt and pepper’ syndrome pa(ent IF iPSC generation and differentiation from S&P fibroblasts
Menendez et al. Proc Natl Acad Sci.108:19240
iPSCs
Dp4
100
90
Relative Abundance
Altered glycolipid 1
profiles in ST3Gal5 cells are more evident in neural crest than in iPSCs 80
Dp3
70
60
50
2+
Gb4
40
30
20
10
0
500
600
700
800
900
Relative Abundance
2
80
1100
1200
1300
m/z
1400
1500
1600
1700
1800
1900
2000
NC cells
Dp4
100
90
1000
Dp3
2+
GD3
2+
GM3
70
2+
GM1
60
50
40
30
20
10
0
500
600
800
700
900
1000
1100
1200
1300
m/z
1400
1500
1600
1700
Relative Abundance
90
80
2000
Dp4
70
60
50
1900
ST3Gal5
iPSCs
100
3
1800
Dp3
2+
Gb4
40
30
20
10
1
2
3
0
500
4
600
700
800
900
1000
1100
1200
1300
m/z
1400
1500
1600
1700
Relative Abundance
90
80
1900
2000
ST3Gal5
NC cells
100
4
1800
Dp4
70
60
2+
GM1b
Dp3
50
40
30
20
10
K. Aoki, M. Tiemeyer
0
500
600
700
800
900
1000
1100
1200
1300
m/z
1400
1500
1600
1700
1800
1900
2000
Differentiation of normal iPSCs to neural crest cells is
accompanied by increased GM3 and more complex gangliosides
Normal iPSCs
Neutral GSLs are abundant.
Normal NC cells
Complex gangliosides.
K. Aoki, M. Tiemeyer
S&P NC cells lack GM3, but have increased GM1b,
GD1c and LacCer
hiC3 NC
NC cells
Dp4
100
90
Dp3
Relative Abundance
80
2+
GM3
70
2+
GD3
2+
GM1
60
50
40
30
20
10
0
500
600
700
800
1000
900
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
m/z
hiC3 NC ST3Gal5
NC cells ST3Gal5
100
90
Relative Abundance
80
Dp4
70
60
Increase of LacCer.
GD1c
ST3Gal2
ST8Sia5
Lack of GM3.
2+
GM1b
Dp3
50
40
30
GM1b
ST3Gal2
20
10
0
500
600
700
800
900
1000
1100
1200
1300
m/z
K. Aoki, M. Tiemeyer
1400
1500
1600
1700
1800
1900
2000
N-linked glycans exhibit cell-specific changes in S&P
neural crest cells
K. Aoki, M. Tiemeyer
Analysis of receptor tyrosine kinases in WT and ST3Gal5
deficient neural crest (NC) cells
WT NC
EGFR
ST3Gal5
ErbB3
WT
InsR IGF-1R
ST3Gal5
ErbB3 ErbB4
WT
EGFR
ST3Gal5 NC
EGFR
ErbB3 ErbB4
InsR IGF-1R
InsR: insulin receptor
EGFR: epidermal growth factor receptor
IGF-1R: insulin growth factor-1 receptor
M. Dookwah, R. Steet
PMM2-CDG and MPI-CDG
Man-P-Dol
Mannose
PMM2
Man-6-P
Man-1-P
MPI
Fru-6-P
GDP-Man
LLO
N-linked glycan
Glu-6-P
CDG Subtype
Gene
Affected
Reaction Affected
PMM2-CDG
PMM2
Man-6-P à Man-1-P
hypotonia, psychomotor retardation,
epilepsy and ataxia, skeletal defects
MPI-CDG
MPI
Fru-6-P à Man-6-P
gastrointestinal and liver problems,
hypoglycemia, thrombotic events
Clinical Features
PMM2 CDG
•  iPSC lines generated from one patient line
-  currently being characterized
PMM2: V231M, R141H mutation
Reduced PMM2 activity in patient human fibroblasts and iPSCs
PMM2: V231M, R141H muta(on 120
PMM activity (%)
100
fibroblast
IPS
80
60
40
20
0
WT
PMM2
Deficient glycoenzyme activities can be detected in cell models
- validates the approach
Noor Klaver and Rich Steet Congenital Disorders of Glycosylation: iPSC Pipeline
Gene Disease Genotypes iPSC Specialized cell types ST3Gal5 Salt and Pepper c.994 G>A (E332K) c.862C>T (nonsense) Yes Neural Crest Neurons PMM2 PMM2-­‐CDG V231M, R141H P113L, R141H F119L, R141H Yes Hepatocyte Neural Crest Neuron POMGnT1 Muscle-­‐Eye-­‐
Brain disease 2 diff. genotypes Yes Cardiomyocytes Neurons OGT X-­‐linked ID 2 diff. genotypes No Neurons COG7 COG7-­‐CDG 2 diff. genotypes No Neural Crest Addi(onal iPSC models under considera(on STT3A/B STT3A/B-­‐CDG ALG9 ALG9-­‐CDG Other hiPSC lines: Congenital muscular dystrophy
(CMD)
Patient/disease
classification
Origin
Cell source
POMGnT1
maturation of α-DG
Wellstone Center
fibroblast
Fukutin CMD
maturation of α-DG
Wellstone Center
fibroblast
FKRP
maturation of α-DG
Wellstone Center
fibroblast
Micheal Tiemeyer (CCRC, UGA)
Kazuhiro Aoki
Rich Steet
Michelle Dookwah
Lance Wells
Michael Pierce
Noor Klaver
Dalton Lab (Center for Molecular Medicine, UGA)
Michael Kulik
Ryan Berger