dopamine neurons derived from human ipscs survive … · dopamine neurons derived from human ipscs...
TRANSCRIPT
DOPAMINE NEURONS DERIVED FROM HUMAN IPSCs SURVIVE AND REVERSE MOTOR ASYMMETRY IN ANIMAL MODELS OF PARKINSON’S DISEASE
Recent studies have indicated that human embryonic stem cells and induced pluripotent stem cells (iPSCs) differentiated into midbrain dopamine (iPSC-mDA) neurons provide functional benefit in animal models of Parkinson’s disease (PD) (Kriks et. al., 2011; Morizane et al., 2013, Grealish et al., 2014, Hallett et al., 2015). Cryopreservation of post-mitotic iPSC-mDA neurons represents a significant vertical advancement for clinical translation of pluripotent stem cell technologies as they are reliably and reproducibly thawed, allowing for rapid access to large numbers of highly pure, autologous cells. In the present study, we examined the engraftment potential of iPSC-mDA neurons after transplantation into the rodent brain. iPSC-mDA neurons were derived from episomally reprogramed human blood samples and cryopreserved in large master cell banks. After thawing, iPSC-mDA neurons retained high viability and maintained gene and protein expression patterns consistent with the midbrain lineage phenotype >1-month in vitro. Patch clamp recordings revealed normal electrophysiological characteristics with firing of evoked and spontaneous action potentials, post-synaptic currents, as well as functional ion channels with characteristic inhibitor responses. In addition, biochemical analysis of iPSC-mDA neurons indicated production of dopamine. Here we demonstrate reversal of motor asymmetry as measured by d-amphetamine- and apomorphine-induced rotations. Immunohistochemical analyses revealed robust long-term graft survival and extensive reinnervation of the host striatum.
iPSC-mDA neurons were cryopreserved on day-38 of differentiation then thawed and plated before RNA isolation. Real-time quantitative PCR was performed using TaqMan Gene Expression Assays. FACS for FoxA2/tyrosine hydroxylase was applied to cryopreserved iPSC-mDA neurons at day-3 post thaw. Immunocytochemistry for FoxA2/Lmx1 and brightfield images for morphology were taken at day-7 post-thaw. Western blots were performed at 4-weeks post-thaw. Dopamine secretion by iCell Dopa Neurons stimulated by HBSS or HBSS with potassium chloride (KCl) was measured by ELISA at 14-days post-thaw. Single- and whole-cell patch clamp recordings were taken at day-21 post-thaw. Female Sprague Dawley rats received medial forebrain bundle (MFB) injections of 6-OHDA. Rats with confirmed behavioral lesions were selected for transplantation (240-310g, 22-23 weeks of age) and immunosuppressed daily with Cyclosporine (10mg/kg, q.24h., i.p.) beginning two days prior to surgery. 3µl (150k/µl) iPSC-mDA were injected unilaterally into the striatum: AP+0.60mm; ML-3.1mm; DV-5.0mm from bregma. MPTP-treated cynomolgus macaques received three injections to the post-commissural putamen bilaterally (150k/µl; 25µl total). Motor asymmetry in rats was assessed by d-amphetamine- (5mg/kg) or apomorphine (0.05mg/kg)-induced rotations every 4-weeks. Rats were sacrificed at 6 months; macaques were sacrificed at 4 weeks (N=2) or 3 months (N=1) via transcardial perfusion (saline followed by 4% PFA). Brains were sectioned coronally and stained for human NCAM, human cytoplasm, human nuclei, ki-67, tyrosine hydroxylase, or triple labeled with FoxA2/TH/human nuclei. Sections were mounted, imaged, and quantified by unbiased stereology.
1. The thaw process yields high purity and viability of midbrain lineage dopamine neurons with functional ion channels and physiologically relevant responses to drug inhibition in vivo.
2. iPSC-mDAs maintain midbrain dopaminergic phenotype in vitro and following transplantation into the rat striatum. 3. Robust graft survival and fiber outgrowth found in both the rat and MPTP monkey brain. 4. Re-innervation of the host rat striatum induced full recovery in d-amphetamine-induced rotations.
1. Assess functional benefit of iCell Dopa Neurons in the behaviorally impaired MPTP-lesioned nonhuman primate model of Parkinson’s disease.
2. Demonstrate long-term safety and functional efficacy with cGMP iPSC-mDA neurons in pre-IND studies. 3. Develop and test functional potential of PD patient-derived iPSC-mDA neurons for autologous transplant in clinical
trial with Rush Movement Disorders clinic.
Fig.1 iPSC-mDA Gene Expression Profile
Fig. 2 Viability and Purity of iPSC-mDA Neurons
Fig. 5 Striatal Graft Survival and Safety After 2 Weeks in vivo
Fig. 6 Maintenance of Midbrain Dopaminergic Phenotype in Grafts In Vivo
Dustin R. Wakeman1 , Benjamin M. Hiller1, David J. Marmion1, Christopher W. McMahon2, Grant T. Corbett1, Junyi Ma2, Jeffrey H. Kordower1 1Department of Neurological Sciences, Rush University Medical Center, 2Cellular Dynamics International Inc.
Figure 1: iCell Dopa Neurons express genes indicative of midbrain floor plate-derived dopaminergic neurons. Markers for forebrain regionalization (FoxG1) or other neuronal subtypes (DBH, vGLUT1, CHAT, OLIG2) are expressed at low levels or negative. Most genes showed very similar expression levels across the time course, up to 42 days post-thaw. The expression profile is similar to human substantia nigra neurons. RT-PCR results are expressed as relative expression to GAPDH control. Values <10-4 are considered background.
Figure 2: (A) Upon thaw, cryopreserved neurons displayed high viability (72% for iCell Dopa Neurons and 81% for iCell Dopa Early Cryo Neurons). (B) FACS analysis showed that iPSC-mDA neurons were 77.1% FoxA2+/TH+ at 14-days post-thaw. (C-C’’) At 7-days post thaw, iPSC neurons exhibit a high percentage of co-localization for midbrain markers FoxA2/Lmx1 and (D) Map2/Lmx1. (E) At 14-days post thaw, iCell Dopa Neurons showed characteristic neuronal morphology with highly branched processes and (F) expression of midbrain dopamine markers FoxA2/TH.
Fig. 7 Functional Analysis of Cryopreserved iPSC-mDA Neurons in Parkinsonian Rats
1.00E-06
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
FO
XG
1
OT
X2
EN
1
FO
XA
2
LM
X1
A
NU
RR
1
TH
AA
DC
GIR
K2
VM
AT
2
DR
D2
DB
H
SN
CA
SY
N1
SY
P
VG
LU
T1
VG
LU
T2
VG
AT
CH
AT
OL
IG2
Rela
tive
Ex
pre
ss
ion
(vs
. G
AP
DH
)
Day 7 PT
day 14 PT
Day 21 PT
Day 28 PT
Day 42 PT
Human SN
Subtype Markers Regional Dopaminergic Neuronal
Day 14 PT
Fig. 8 Three Month iPSC-mDA Graft Survival in MPTP-Treated Macaques
Figure 8: (A, B) iCell Dopa Neurons transplanted into the striatum of behaviorally asymptomatic MPTP-treated cynomolgus macaques survived and projected fibers into the host striatum after 3-months. (C,D) huCyto+ cells developed characteristic neuronal morphology and displayed robust fiber outgrowth in (C, D) iCell Dopa Neuron grafts. (E, F) iCell Dopa Neurons injected into the putamen had TH+ cell bodies and fibers at 3-months post-transplant. (G, H) Compared to 1-month post-grafting, TH+ cell bodies had a more mature morphology with larger, arborized soma. In addition, (H) TH+ engrafted cells extended fibers out from the body of the graft and innervated host tissue. Scale bars represent 250µm (B, F, G), 500µm (A), 1000µm (E), 50µm (H inset), or 25µm (C, D, H).
Figure 4: (A) iCell Dopa Neurons secreted dopamine in vitro when cultured in HBSS or when challenged with 56mM KCl induced depolarization, while iCell Neurons (GABAergic forebrain lineage) do not. (B) iCell Dopa Neurons fired spontaneous action potentials (4 of 6 recorded cells). (C) Action potentials were evoked in response to current injection (6 of 6 recorded cells). (D) Na+ channels respond to TTX inhibition in a dose-dependent manner and (E) K+ channels are inhibited by TEA, indicating functional ion channels with characteristic inhibitor responses. All electrophysiology recordings took place at 21-days post thaw.
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
iCell DopaNeurons iCell Neurons
Do
pam
ine
Co
nce
ntr
atio
n (
ng/
mL)
HBSS
KCl stim
Samples
were run in
triplicate
A
-80 -60 -40 -20 0 20 40 60
-150
-100
-50
0
Cu
rren
t d
en
sit
y (
pA
/pF
)
Voltage (mV)
10 100
0.0
0.2
0.4
0.6
0.8
1.0
% o
f cu
rren
t
TTX (nM)
IC50=11.9 nM
500 pA
1 ms
A B CD
TEA 30 mMControl
200 pA
100 ms
-80 -60 -40 -20 0 20 40 60
0
20
40
Cu
rren
t d
en
sit
y (
pA
/pF
)
Voltage (mV)
Control
TEA 30 mM
N=5
A B CE
A
10 mV
1 s
0 mV
B
10 mV
100 ms
B
Figure 5: (A) iCell Dopa Neurons projected fibers in the striatum of immunosuppressed Sprague-Dawley rats and (B) retained expression of Tyrosine Hydroxylase (TH) at 2 weeks post-transplantation. (C) Staining for human Nuclei demonstrates the size and density of iPSC-mDA striatal grafts at 2 weeks post-transplantation. In level matched sections, no Ki-67+ cell were found in any grafts. (B’’, D’’) Endogenous Ki-67+ cells were located in the subventricular zone (SVZ) as expected. (E) Table shows the mean survival and coefficient of error, as well as the standard error of the mean of human Nuclei+ cells/hemisphere surviving after 2 weeks in vivo. C.E. listed as Gunderson m=1. Scale bars represent 25µm (D’, D’’), 50µm (B), 250µm (A), or 500µm (C, D).
E E’
human nuclei/FoxA2/TH
FoxA2/Hoechst
94% FoxA2+
Lmx1/Hoechst
96% Lmx1+
FoxA2/Lmx1
91% FoxA2+/Lmx1+
Day
7 P
ost
-th
aw
C C’ C’’
0%
20%
40%
60%
80%
100%
iCell DopaNeurons iCell Dopa Early Cryo
% V
iab
ilit
y (
Try
pan
Blu
e)
Thaw Viability A
Map2/Lmx1
FoxA2/TH/Hoechst
B
Isotype Day 14 Post-Thaw
E F
TH-AF488
Fo
xA
2-A
F6
47
human NCAM A TH B
Figure 3: Cryopreserved iCell Dopa Neurons (iPSC-mDA) were cultured for 4-weeks then analyzed for expression of neuronal proteins. Western blot analysis confirmed an expression profile consistent with the mature midbrain dopaminergic lineage compared to control iPSC-derived forebrain GABAergic neurons (iPSC-GABA) or human fetal cortical neural stem cells cultured in dopaminergic medium (NSC-DA).
This study is supported by grants from the Rush Translational Sciences Consortium and Cellular Dynamics International Inc./Fuji Film to DRW. Special thanks to Yinzhen He, Yaping Chu, Katie Nice, Gina Mazzei, Scott Muller, and Rachel Harker for technical assistance, as well as J.P. Werneck De Castro for statistical expertise and Tomas Bjorklund and Roger Barker for procedural advice.
Fig. 3 Biochemical Analysis of iPSC-mDA Neurons
D
Brightfield
E
Day
14
Po
st-t
haw
GABAergic
GABRA2
GAD65/67
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
Glutamatergic
VGlut1
VGlut2
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
Cholinergic
ChAT
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
VMAT2
Dopaminergic
TH
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
GIRK2
Calbindin
Neuronal
Synapsin-1
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
PSD-95
MAP2
GAP43
DCX
Development
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
Nestin
FoxA2
EN1
PITX3
iPS
C-m
DA
iPS
C-G
AB
A
NS
C-D
A
Midbrain Floor Plate
FoxA2 human nuclei TH TH TH TH TH
human cytoplasm human cytoplasm human cytoplasm A
E G F
D C human cytoplasm
Figure 7: Co-localization of FoxA2+/TH+/huNuclei+ neurons after 2-weeks post-transplantation in the rat striatum demonstrated maintenance of the midbrain dopaminergic lineage in iCell Dopa Neuron grafts. TH+ (green) cell bodies projected processes, innervating host tissue at 2-weeks post-transplantation. Many huNuclei+ (blue) cells co-localized with FoxA2+ (red) cells, indicating midbrain lineage for a large fraction of the cell population. Scale bars represent 25µm.
H
B
Conclusions
Future Directions
Funding & Acknowledgements
Introduction
Methods
Fig. 4 DA Release and Electrophysiological Activity of iPSC-mDA Neurons
huNuc
lei C human nuclei
V
cc
D
D’’
D’
Ki-67 Cell Survival
Mean (Survival %)
24.08
Standard Error (%)
8.00
Mean C. E.
0.06
Grealish S, Diguet E, Kirkeby A, Mattsson B, Heuer A, Bramoulle Y, Van Camp N, Perrier AL, Hantraye P, Björklund A, Parmar M. Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease. Cell Stem Cell. 2014 Nov 6;15(5):653-65. Hallett PJ, Deleidi M, Astradsson A, Smith GA, Cooper O, Osborn TM, Sundberg M, Moore MA, Perez-Torres E, Brownell AL, Schumacher JM, Spealman RD, Isacson O. Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease. Cell Stem Cell. 2015 Mar 5;16(3):269-74. Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang L, Beal MF, Surmeier DJ, Kordower JH, Tabar V, Studer L. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature. 2011 Nov 6;480(7378):547-51. Morizane A, Doi D, Kikuchi T, Okita K, Hotta A, Kawasaki T, Hayashi T, Onoe H, Shiina T, Yamanaka S, Takahashi J. Direct comparison of autologous and allogeneic transplantation of iPSC-derived neural cells in the brain of a non-human primate. Stem Cell Reports. 2013 Sep 26;1(4):283-92.
References
Figure 7: Adult Sprague-Dawley rats were unilaterally lesioned with 6-OHDA into the right medial forebrain bundle to create a stable dopaminergic lesion. Only animals that exhibited sustained rotational asymmetry as confirmed via amphetamine-induced rotations were used to assess functional output. 8-weeks post-lesion, cryopreserved iPSC-mDA neurons or vehicle control were transplanted into the ipsilateral striatum of CsA immunosuppressed rats. At 6-months post-transplantation, histological analysis revealed unilateral degeneration of TH+ midbrain dopaminergic neurons (A,E) and their striatal projections (B,J). Animals grafted with iPSC-mDA neurons showed robust graft survival with extensive innervation of Human NCAM+ fibers into the engrafted striatum (C). Human Nuclei+ cell bodies (D) remained within the injection site and did not migrate into peripheral brain structures. Transplanted iPSC-mDA TH-ir neurons (F-J) innervated the entire striatum, projecting fibers caudally into ventral structures without proliferation (K). Critically, all animals with successful grafts (N=6) demonstrated statistically significant reversal in amphetamine and apomorphine-induced rotational asymmetry (L,M) compared to animals with unsuccessful grafts (N=4) or vehicle controls (N=4) indicating both dopamine release from grafted iPSC-mDA neurons, as well as normalization of D2 receptors secondary to innervation. Abbreviations: ac (anterior commissure), acp (anterior commissure anterior), cc (corpus callosum), CPu (Caudate Putamen), GP (Globus Pallidus), LV (Lateral Ventricle). Statistical analyses: A two-way ANOVA was performed using Bonferroni’s post-hoc test for multiple comparisons.
C
E
-1 1 2 3 4 5 6
-5
0
5
10
15
20
25
30
Months
Ro
tati
on
s / M
inu
te
Amphetamine Rotations
(+) Graft (-) Graft Vehicle
Transplant
*********
********
** = 0.0048 *** = 0.0004 **** < 0.0001****
-1 1.5 3 4.5 6-20
-15
-10
-5
0
Months
Ro
tati
on
s / M
inu
te
Apomorphine Rotations
(+) Graft (-) Graft Vehicle
Transplant
*
#
*
* = 0.0351 * = 0.0170 # = 0.0191
TH TH TH TH
Ki-67 TH
A B
6-OHDA Control 6-OHDA iCell Dopa
human NCAM human Nuclei
6-O
HD
A i
Ce
ll D
op
a
TH TH
6-O
HD
A i
Ce
ll D
op
a
6-O
HD
A C
on
tro
l
C D
E F G H
I J
TH
aca
CPu
cc
ec
LV LV CPu
cc
ec
aca
LV
L
M
K