Read a paper, read a story

Chao Liu Ph.D.

Department of Histology and Embryology, Institute

of Stem cell and Tissue Engineering, School of
Basic Medical Sciences
Anhui Medical University
2011.11.28
 Where and how you get papers?
• From internet sourses:
Pubmed, Web of science,…

• According to key words:

your research interests, grants,…
Example:
Parkinson’s disease

A slowly progressive most common movement disorder.

 Pathology of PD

Midbrain crosssection
Selective loss of dopaminergic neurons in the
substantia nigra.
AHMU 4
 Stem cell therapy for PD

Normal Neurodegeneration Neuroregeneration

J. Clin. Invest. 120:29–40 (2010).

02/08/2024 5
 Question?
• Where you get enough dopaminergic
neurons?
 Directed Differentiation:
from human ESCs/hIPSCs
to midbrain dopamine (mDA) neurons

1. Differentiation of human ESCs/iPSCs into mDA

progenitors.
2. Differentiation of mDA progenitors into mDA
mature neuron.

(Ferri A L M et al. Development,

2007)
AHMU 7
Research related to my interest
 3. Methods
1.Title

2.Abstract
 Key points of the title

• Purification
• Human ES
• IPSC
• Midbrain dopaminergic (mDA) progenitor
• LRTM1
The other key points of the Abstract/summary

• Parkinson’s disease(PD)
• Cell sorting
• PD animal model
• Cell transplantation therapy
 Abstract
•Human induced pluripotent stem cells (iPSCs) can provide a promising source of
midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson’s
disease (PD).
•
•However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells.

•To eliminate these unwanted cells, cell sorting using antibodies for specific markers such
as CORIN or ALCAM has been developed, but neither marker is specific for ventral
midbrain.

•Here we employ a double selection strategy for cells expressing both CORIN and
LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When
transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1t cells survive and
differentiate into mDA neurons in vivo, resulting in a significant improvement in motor
behaviour without tumour formation. In addition, there was marked survival of mDA
neurons following transplantation of LRTM1t cells into the brain of an MPTP-treated
monkey.

•Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.
 Key points of the methods

• Production of LMX1A::GFP KI mESC line.

• Maintenance and neural differentiation of
mESCs/iPSCs.
• Maintenance and neural differentiation of
hESCs/iPSCs.
• Transplantation into the rat PD models.
Transplantation into the non-human primate PD
models.
• Dopamine release assay.
• Electrophysiological analysis.
• Microarray analysis.
 Sumarry and conclusions

• Parkinson’s disease(PD) /PD animal model

• Cell transplantation/Cell replacement therapy
• Midbrain dopaminergic (mDA) progenitor
• Cell sorting/Purification
• Human ES
• IPSC
• LRTM1

Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.
 Schematic representation

How the authors draw a conclusion?

Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.

Differentiation Cell sorting Cell

Human ES PD
of mDA of mDA replacement
Human iPSCs model
progenitors progenitors therapy
 Research areas

• Parkinson’s disease (PD)

• Human ES: embryonic stem cells

• Human iPSC : human induced

pluripotent stem cells
 Technique Backgrounds

• Production of LMX1A::GFP KI mESC line.

• Maintenance and neural differentiation of
mESCs/iPSCs.
• Maintenance and neural differentiation of hESCs/iPSCs.
• Transplantation into the rat PD models. Transplantation
into the non-human primate PD models.
• Dopamine release assay.
• Electrophysiological analysis.
• Microarray analysis.
• …
 What is a stem cell?

Self-renewal Differentiation
stem cell
(copying) (specializing)

stem cell specialized cells

AHMU 18
 Where are embryonic stem cells?

Tissue/adult
stem cells
Human embryonic stem cells fetus, baby and
(hESCs) throughout life

Induced pluripotent stem cells (iPSCs)

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 Human Embryonic Stem Cells

“These cell lines should be useful in

James Thomson. human developmental biology, drug
University of Wisconsin . discovery, and transplantation
medicine.”

Human embryonic stem cell (hESC)

(Thomson JA, et al. Science, 1998)

 Induced Pluripotent Stem Sells (iPSCs)

Reprogramming
Shinya Yamanaka Nobel prize!

Induced Pluripotent Stem Sells (iPSCs)

(Takahashi and Yamanaka.2007).

AHMU 21
 Reprogramming
• Reprogramming by four factors: Oct4, Sox2, Klf4 and c-Myc

I. Preparation of human II. Infecting III. Picking up ES-like IV. Passaging

skin fibroblasts fibroblasts colonies Cryopreserving

(Takahashi and Yamanaka.2007).

 Promises of stem cell research

Drug
discovery

AHMU 23
02/08/2024 23
 Promises of iPSC

Patients iPSCs
(with gene mutation)

Disease
Adult cells
modeling

Drug Cell
screening therapy

Virus infection (modified from Passier R ,et al.2008; Evangelos Kiskinis and Kevin Eggan, 2010)
02/08/2024 24
 Why iPSCs?

Better than animal models

• Develop new human disease models.
• For the study of the mechanisms of
human diseases…
 Question?
• How you get the cells?
 Neuronal differentiation

(Broccoli etal. Frontiers in neuroscience, 2014)

02/08/2024
 Neurodevelopment
Neuroectoderm: primordium of nervous system

Neuroectoderm
?
?
(Modified from Journal of Cellular Biochemistry
4, 795-805 (October 2003) 105:633–640 (2008)) 28
02/08/2024 28
 Neural development and neurogenesis

Neural
Neurogenesis
development
 To present a powerful system
for the in vitro dissection of
human neurogenesis.
Front Mol Neurosci 4 (2011) 30

（ Petros T.J. et al. Frontiers in molecular neuroscience 2011 ）

02/08/2024 29
What we are doing?
 Improved neurogenesis

A H9 Stage I Stage II

d0 d5, d6 d10, d11

On matrigel On matrigel On matrigel

in mTeSRTM1 in NE induction medium In neuronal
induction medium

B d5, beta-III-tubulin/DAPI C d11, beta-III-tubulin/DAPI D Day 0 5 10

KDa
OCT4
42
PAX6 51

beta-III
-tubulin 51
GAPDH
29
1 2 3

Beta-III-tubulin/Tuj-1:
early neuron marker
(Liu, et al. BBRC, 2014.)

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 Localized high cell density
promotes NE induction

PAX6 ： neuroectoderm marker

Nestin: neural stem cell marker
(Liu, et al. BBRC, 2014.)

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 SMAD signaling blockade promotes
NE Induction

(Liu, et al. BBRC, 2014. )

02/08/2024 33
A simple method for NE induction

(Liu a, et al, Tissue&Cell, 2015 )

From NE to
dopaminergic
neurons.

DA neuron: TH positive

(Liu a, et al, Tissue&Cell, 2015 )

 Midbrain dopaminergic neuron

NE induction DA neuron

Midbrain DA neuron: TH positive and Pitx3 positive

More markers are better.

02/08/2024 36
What’s in this paper?
 Figure 1. Purification of mDA
progenitors by co-expression of
CORIN and LMX1A::GFP.
 In vivo data: a nature

(a)Schematic
Midbrain
diagram of
CORIN and
Forebrain Hindbrain LMX1A
expression
during early
development of
mouse.
(b,c)
Immunohistochemical
images for CORIN
(green) and LMX1A
(red) in coronal and
sagittal sections of
E11.5 fetal mouse.
Scale bars, 50 mm (b)
and 200 mm (c).
Mouse ESC differentiation

(d) Schematic diagram of

neuronal differentiation from
mESCs.

(e,f) LMX1A::GFP expression of

the serum-free floating culture of
embryoid body-like aggregates
with the quick reaggregation
(SFEBq)-cultured mESC
aggregates on day 2 (e) and
day 9 (f).
 mDA generation
(g–o)
Immunofluorescence
images of the cells from
unsorted and
CORIN+LMX1A::GFP+
cells for TUJ1 (green),
NURR1 (green), DAT
(green), TH (red) and 40,
60-diamidino-2-
phenylindole (DAPI;
blue) on day 14. Scale
bars, 70 mm.
Quantification
of TUJ1+TH+, NURR1+TH+
and DAT+TH+ cells in
unsorted cells (n=4)
versus
Tuj1: neuron marker protein CORIN+LMX1A::GFP+
TH, NURR1, DAT: mDA marker proteins cells (n=4)
on day 14.
 Statistic analysis

Asterisks indicate
statistical significance as determined by Student’s t-test,
*P<0.05 and ***P<0.001. Error bars indicate s.e.m.
 Summary
• These results indicate that
mDA progenitors were enriched in the
CORIN+LMX1A::GFP+ population.
Figure 2. Gene expression profiles between CORIN +
and CORIN- populations from mESC-derived
LMX1A::GFP+ cells or fetal mouse VM.
(a) Comparison of gene expression profiles between CORIN+ and
CORIN-cells in LMX1A::GFPt+ cells on day 9. Scale bars, 250
mm.
(b) Comparison of gene expression profiles between CORIN+ and
CORIN- cells in E11.5 fetal mouse VM. Scales bars, 50 mm.
(c) Semi-quantitative RT–PCR analysis of Otx2,Corin, Anxa2,
Tm4sf1, Folr1, Tacr1, Lrtm1 and Gapdh in E11.5 fetal mouse brain.
 Summary
• LRTM1 is a cell surface marker for mDA
progenitors.
 Figure 3. LRTM1 is selectively
expressed in VM during early brain
development.
(a) Schematic construction of LRTM1. (b) In E11.5 fetal mouse,
LRTM1 mRNA was detected in the brain, eye, lung and heart. (c)
In adult mouse, LRTM1 mRNA was detected in the eye and heart
only.
 Figure 3. continued

LMX1A: ventral
midbrain marker

(d–o) Immunohistochemical images of fetal mouse midbrain for 40,

60-diamidino-2-phenylindole (DAPI; blue), LMX1A (red) and LRTM1
(green). Insets indicate magnified images of LMX1A+ DA
progenitors. Scale bars, 150 mm.
 Figure 3. continued

FOXA2: ventral
midbrain DA
marker

(p) Immunohistochemical image of E10.5 fetal mouse for LRTM1 (green),

LMX1A (red) and DAPI (blue) in the sagittal section. Inset indicates magnified
images of LMX1At DA progenitors. Scale bar, 400 mm.
(q) Immunohistochemical image of E10.5 fetal mouse for LRTM1 (green),
FOXA2 (red) and DAPI (blue) in the coronal section. Inset indicates magnified
images of FOXA2+ FP cells. Scale bar, 150 mm.
 Figure 3. continued

(r) Quantification of LRTM1t cells in E11.5 VM (n=4) versus E13.5 VM

(n=4). (s) Quantification of immunoreactive cells in the hindbrain stained
for anti-Corin (n=4) versus anti-LRTM1 antibodies (n=4). Asterisks
indicate statistical significance as determined by Student’s t-test,
*P<0.05 and **P<0.01. Error bars indicate s.e.m.
 Summary
• These results indicate that the expression
of LRTM1 in the developing brain is
restricted in terms of the developmental
stage, the time of emergence of mDA
progenitors and location, namely the VM.

LRTM1 mDA progenitors

Figure 4. Human LRTM1+ cells generate
mature mDA neurons in vitro.
(a) Diagram of neuronal differentiation from
human PSCs (hPSCs).
 Figure 4. continued

(b) Immunofluorescence image of a hESC-derived LRTM1+ sphere on

day 14 for FOXA2 (green), LMX1A (red) and 40, 60-diamidino-2-
phenylindole (DAPI; blue). Scale bars, 100 mm. (c) Quantification of
FOXA2+LMX1A+ cells in unsorted cells versus LRTM1+ cells on day 14
(hESC: n=6; hiPSC: n=6).
 Figure 4. continued

(d–f) Immunofluorescence images of spheres from unsorted

cells, LRTM1+ cells and LRTM1 cells on day 28 for NURR1
(green), TH (red), FOXA2
(white) and DAPI (blue). Scale bars, 100 mm.
 Figure 4. continued

(g) Immunofluorescence images of a sphere from LRTM1t cells for FOXA2

(green), NURR1 (green), TH (red) and DAPI (blue) on day 28. Scale bars, 50
mm. Quantification of TH+FOXA2+ (h) and TH+NURR1+ cells (i) in unsorted
cells (hESC: n=4; hiPSC: n=3) versus LRTM1+ cells (hESC: n=4; hiPSC: n=3)
versus LRTM1 cells (hESC: n=3; hiPSC: n=3).
A one-way analysis of variance with Bonferroni’s multiple comparison test,
*P<0.05, **P<0.01 and ***P<0.001 (h,i,m–p,u). Error bars indicate s.e.m.
 Figure 4. continued

(j–l) Immunofluorescence images of spheres from

unsorted and LRTM1+ cells for NESTIN (green), SOX1 (green), TUJ1 (red),
PAX6 (red), 5-HT (red), KI67 (white) and DAPI (blue) on day 28. Scale bars,
50 mm.
 Figure 4. continued

Quantification of NESTIN+ (m), TUJ1+ (n), KI67+ (o), SOX1+PAX6+ (p) and 5-HT+ cells
(q) in unsorted cells (hESC: n=5; hiPSC: n=4) versus
LRTM1+ cells (hESC: n=5; hiPSC: n=4) versus LRTM1 cells (hESC: n=5; hiPSC: n=4).
A one-way analysis of
variance with Bonferroni’s multiple comparison test, *P<0.05, **P<0.01 and
***P<0.001 (h,i,m–p,u). Error bars indicate s.e.m.
 Figure 4. continued

(r,s) Immunofluorescence images of LRTM1+ cells for PITX3

(green), DAT (green), TH (red) and DAPI (blue) on day 70. Scale bars, 10 mm. (t)
Current clamp recordings of induced action potentials by brief current
pulses from human ES-derived DA neurons on day 70. (u) Levels of DA in hESC-
derived unsorted (n=5) versus LRTM1+ (n=5) versus LRTM1 (n=5)
cultures on day 42. Asterisks indicate statistical significance as determined by
Student’s t-test, *P<0.05 and ***P<0.001 (c), and a one-way analysis of
variance with Bonferroni’s multiple comparison test, *P<0.05, **P<0.01 and
***P<0.001 (h,i,m–p,u). Error bars indicate s.e.m.
 Summary
• Enrichment of human mDA progenitors
and mature neurons by LRTM1 sorting.
Figure 5. Human LRTM1+ cells generate functional
DA neurons in vivo following transplantation.
(a–c) Immunofluorescence images of the
graft for SC-121 (green) and TH (red) at 12
weeks. Scale bars, 1 mm.
(d,e) Quantification of graft volumes and HNA +
cells in unsorted cells (n=7) versus
LRTM1+ cells (n=12) versus LRTM1 cells
(n=5) at 12 weeks.
Immunofluorescence images of a graft containing unsorted cells (f)
and LRTM1+cells (g) for 40, 60-diamidino-2-phenylindole (DAPI;
blue), HNA (green) and TH (red) at 12 weeks. Scale bars, 100
mm. (h–l) Immunofluorescence images of a graft containing
LRTM1+ cells for GIRK2 (green), CALBINDIN (green), FOXA2
(green), NURR1 (green), HNA (green), TH (red), KI67 (red) and
DAPI
(blue) at 12 weeks. Scale bars, 25 mm.
Quantification of TH+ (m), TH+HNA+ (n), FOXA2+HNA+ (o),
NURR1+HNA+ (p) and KI67+HNA+ (q) cells in
unsorted cells (n=7) versus LRTM1t cells (n=12) versus LRTM1 cells
(n=5) at 12 weeks. Asterisks indicate statistical significance as determined
by a
one-way analysis of variance (ANOVA) with Bonferroni’s multiple
comparison test, *Po0.05, **Po0.01 and ***Po0.001. Error bars indicate
s.e.m.
(r,s) Quantification of motor behavior of 6-OHDA-lesioned rats for 16 weeks
posttransplantation. (r) Methamphetamine-induced rotation (control: n=8;
unsorted: n=7; LRTM1t: n=7) was performed every 4 weeks after
transplantation. (s) Apomorphine-induced rotation was performed every 8
weeks after transplantation (control: n=7; unsorted: n=7; LRTM1t: n=7).
Asterisks indicate statistical significance between control and LRTM1+ as
determined by a two-way ANOVA with Bonferroni’s multiple comparison test,
*Po0.05 and ***Po0.001. Dagger indicates statistical significance between
control and unsorted as determined by a two-way ANOVA with Bonferroni’s
multiple comparison test, wPo0.05.
 Summary
• Human LRTM1+ cells differentiated into
mDA neurons in vivo.
 +
Figure 6. HiPSC-derived LRTM1 cells
survived and differentiated into mature
DA neurons in primate PD model.

How to prove?

Your home work

Thank you!