Summary 10 yrs protocol for developing neural cells from Zhang et al., articles

1. Differentiation of neural cells
in human and mouse
embryonic stem cells (ESCs)
Presenter: Han-Ni Cheng
20120919
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2. Outline
 Embryonic stem cells (ESCs)
 Inducing neural differentiation from
hESC.
 The linkage between neural cells and
retinal cells
 Discussion
2

3. Embryonic stem cells
(ESCs)
3

4. ESCs
 Evans established mouse embryonic stem
cells in 1981.
 Reynolds and Weiss cultured neural stem
cells as neurospheres in vitro in 1992.
 ES cell lines derived from human blastocysts
by Thomson et al. in 1998.
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5. 5
ESCs
• Infinite source of replacement parts.
• Compatibility: No organ rejections.
• Insights into complexity of signaling
during human development.
• Testing of new drugs.
• Cell-based therapies.

6. 6
•hESC culture: usually using H9 and H11 lines•hESC culture: usually using H9 and H11 lines•hESC culture: usually using H9 and H11 lines
hESC lines ex: H9 and H11 lines
1. maintain culture medium:
•maintain culture medium:
Chemical Final concentration
DMEM/F12 (Gibco, Rockville) Basic medium (1:1)
knockout serum replacement (Gibco) 20% (v/v)
L-glutamine (Sigma, St. Louis) 1 mM
NEAA (Gibco) 0.1 mM
β-Mercaptoethanol (Sigma) 0.1 Mm
FGF2 (R&D Systems, Minneapolis) 4 ng/ml
p.s. cultured on a feeder layer of mouse embryonic fibroblasts (MEF) with a
daily change medium. 37°C and under an atmosphere containing 5% of CO2.
(Sebastien et al., 2008; Zhang et al., 2005; Zhang et al., 2010)(Sebastien et al., 2008; Zhang et al., 2005; Zhang et al., 2010)(Sebastien et al., 2008; Zhang et al., 2005; Zhang et al., 2010)
(Sebastien et al., 2008; Zhang et al., 2005; Zhang et al., 2010)

7. 2. Subculture (passage):
• Passage weekly
• Removing hES colonies intact from feeder layer
- 0.1-0.2 mg/mL dispase (Gibco) at 37°C for 30 min
(Zhang et al., 2001)
- 1 mg/mL dispase (Gibco) at 37°C for 3-5 min (Hu
B. Y. and S. C. Zhang, 2009)
7

8. Differentiation of neural cells
from ESC in Dr. Su Lab
system
8

9. 9
Hb9-eGFP Sox1-eGFP
Non - feeder support feeder culture
• Maintain: 10 % KSR with 1 %
FBS and LIF.
• Differentiation: 10 % KSR
without FBS and LIF.
Mouse system
Density: 1~2
*105 cells/ mL

10. Inducing neural differentiation from
hESC in vitro
10
ES
(ref. Gaspard N. and Pierre V., 2009)
rosette formation Single cell
from rosette
Fig. 1. Temporal patterning during ES cell
neurogenesis
Neural
stem
cells

11. 11Fig. 2. Neuron Types. (Tobin T. 2003)
Neurons

12. 12
Astrocytes also called “star cells”
 Inducing endothelial cells to form tight junction (in brain).
 Provision of nutrients to the nervous tissue.
 Maintenance of extracellular ion balance. Most of calcium.
 Playing a role in the repair and scarring process of the
brain and spinal cord following traumatic injuries.
 Many of these cells express the intermediate filament glial
fibrillary acidic protein (GFAP)

13. 13
 Their main functions are to provide support and to
insulate the axons (the long projection of nerve cells) in
the central nervous system (the brain and spinal cord) of
some vertebrates. (The same function is performed by
Schwann cells in the peripheral nervous system).
Oligodendrocytes

14. 14
Type Time Authors Journal
neural precursors 2001 Zhang et al. Nature
Dopaminergic
neuronal subtypes
2005 Yan et al. Stem Cells
oligodendrocytes 2009 Hu et al. Development
Spinal motor
neurons
2009
Hu B. Y. and S. C.
Zhang
Nature
neuroectoderm cell 2010 Zhang et al. Cell Stem Cell
astroglial subtypes 2011 Robert et al. Nat. Biotechnol.
neurons 2011 Jason et al. PNAS
Table 1. Inducing neural differentiation from hESC.

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Differentiation of neural precursors
hESC colonies Growing in suspension as
embryoid bodies for 4 days
Cultured in the a tissue-treat
flask in neurosphere medium
containing FGF2 for 5 days
smalll, elongated cell
Dispase
treatment
By 7 days, the central cells had
generated rosette formation
The
Fig. 1-1. Schematic diagram of human ES cell differentiation into types of
neural cells. Scale bars = 100 μm. (Zhang et al., 2000; Zhang et al., 2001)

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Fig. 1-2. Schematic diagram of human ES cell differentiation into types
of neural cells. Scale bars = 100 μm.
Dispase treatment
Culture on a coating with
ornithine and laminin substrate
Spilt every other week
Maintaining 8 passages
Identifications
 Immunocytochemical staining (ICC)
 Electrophysiological Recording
(Zhang et al., 2000; Zhang et al., 2001)

17. 17
Table 2-1. Identification of neural cells by ICC.
Cell type Markers
Rosette cells Nestin and Musashi-1
Neurons (including
neurotransmitters)
Tyrosine hydrolase , βIII-tublin and MAP2
Oligodendrocytes
O4, Platelet-derived growth factor - α (PDGFR
- α) and MBP.
Astrocytes GFAP, NF200 and S100 β
Neuroepithelial
(NE)
OTX2
(ref. form Table 1)

18. 18
Table 2-2. Identification of neural cells by ICC.
Marker Cell Type
NF-L Neurons
NF-M Neurons
NF-H Neurons
Peripherin Neurons
Α-Internexin Neurons
Vimentin Glia cells, astocytes
Nestin Glia cells, astocytes, neuroepithelial cells
GFAP Glia cells, astocytes
(Perspectives of Stem Cells, Ulrich H., 2010)

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Fig. 2. Cells within a cluster of rosettes (lower left) and a small evolving
rosette (center) are positive for Nestin and Musashi-1.
Nestin Musashi-1 Merge
Rosette cells
(Zhang et al., 2001)

20. 20
A B
Fig. 3-1. Identification of astrocytes. (A) Differentiation of a
cluster of ES cell–derived neural precursors for three. (B)
Immunostaining after three weeks of differentiation indicates
that the majority of cells are βIII-tubulin+ neurons (red) and
that only a few cells are GFAP+ astrocytes (green). Scale bars =
100 μm.
Astrocytes
(Zhang et al., 2001)

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Neurons (including neurotransmitters)
Glutamate GABA Tyrosine hydroxylase
O4+ oligodendrocytes (arrows)
Fig. 3-2. Identification of neurons.
(Zhang et al., 2001)

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Neural differentiation
•Mouse model:
Day 0 Day 2 Day 9-10
neurobasal medium
with 2 ng/mL of LIF
neurobasal medium
Neuroepithelial aggregates
attached

23. Neurobasal medium for mouse ESCs
23
Chemical Final Concentration
DMEM/F12 (Gibco, Rockville) Basic medium
β-Mercaptoethanol (Invitrogen) 0.1 mM
L-glutamine 1 mM
N2 neural supplement 1 X
Lipid concentrate 1 X
N-acetyl cysteine 40 μg/ml
(Zhang et al., 2010)

24. 24
Day 0 Day 4 Day 7
hESCM with
no FGF2
neural differentiation
medium
Day 10 Day 15 Day 28 Day 35
neural
differentiation
medium with
RA 0.1 μM
EBs attached
• neural differentiation medium with RA 0.1 μM, B 27 and SHH 100 ng/mL
• neural differentiation medium with BDNF, GDNF and IGF1 10 ng/mL, cAMP 1
μM, ascorbic acid 200 ng/mL, SHH 50 ng/mL and RA 50 nM.
Isolation

25. 25
Neural differentiation medium for
human ESCs
(Hu B. Y. and S. C. Zhang, 2009)
Chemical Final volume (mL)
DMEM/F12 (Gibco, Rockville) 489
β-Mercaptoethanol (Invitrogen) 1:100
Heperin (Sigma) 1 mL, 1 mg/mL
N2 neural supplement 5

26. 26
Day 0 Day 4 Day 8-10
hESCM with
no FGF2
neural precursor cells medium
EBs attached
Isolation of neural
precursor cells
neural precursor cells medium
with FGF2 (20 ng/mL)

27. Neural precursor cells medium
(neurosphere medium) for human ESCs
27
Chemical Final Concentration
DMEM/F12 (Gibco, Rockville) Basic medium
Insulin 25 μg/ml
transferrin 100 μg/ml
Heperin (Sigma) 2 μg/ml
progesterone 20 nM
EGF (Collaborative Biomedical Products) 20 ng/mL
FGF2 (Collaborative Biomedical Products) 20 ng/mL
LIF (R & D System) 20 ng/mL
(Zhang et al., 2000)

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29. 29

30. Dopamine
30
Day 0 Day 4 Day 8-10
hESCM with
no FGF2
neural cells medium
EBs attached
Isolation of neural
precursor cells
neural precursor cells medium
with FGF2 (20 ng/mL)

31. 31
Chemical Final volume (mL)
DMEM/F12 (Gibco, Rockville) 489
β-Mercaptoethanol (Invitrogen) 1:100
Heperin (Sigma) 1 mL, 1 mg/mL
N2 neural supplement 5

32. 32
• According Hu et al. (2009), FGF2 regulate the early
stage of neural development, and later stage be
determine by retinoic acid (RA) and sonic hedgehog (SHH).
• FGFs signal is a upstream MEK/ERK, PIK3/AKT pathway.

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The linkage between neural
cells and retinal cells

34. Retina
• The mammalian neural
retinal is derived from
retinal progenitor cells
(RPC) of neuroectodermal
origin, initially found in
the protrusive optic
vesicles on both sides of
neural tube.
34
(Perspectives of Stem Cell, Henning
Ulrich, 2010)
(Development Biology Online, Sandra K. Ackerley, 2012)

35. 35
(Mitchell K., 2010)Fig. 4. The mature retina structure.

36. Conclusion
• Retina is a special structure and there has different
neural cell types but didn’t including in common
classification of neurons.
• There have stable differentiating into neural cells
system in mouse, chick and human ESCs, but
retinal cells still criticized.
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37. The End
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38. Neurogenesis in vivo
38
•自受精day 1算起
- day 16: 脊索開始形成，胚胎開始進行養分消化。
- day 20: 神經核即將形成腦囊，此時胚中血管開始分布。
- day 22: 神經摺始融合；心肌形成有脈搏；肝臟雛型，肺臟原型形成；視神經溝出現。
- day 24: 視神經囊與眼窩形成。
- day 26: 上肢開始突出。
- day 28: 水晶體出現；脾臟與泌尿道出現。
- day 32: 眼球形成；排泄與生殖管道出現。
- day 35: 眼杯內層為視網膜細胞，外層為視網膜感光上皮細胞。
- day 37: 視網膜色素產生。
- day 44: 骨骼開始成形。
- day 47: 泌尿、生殖隔膜裂開。
- day 54: 耳部、眼瞼發育更趨成熟。
- day 58: 實心視神經 (optic nerve)，源自第二對腦神經。
(Larsen 人類胚胎學 第二版)

39. 39
Temporal patterning during ES cell
neurogenesis.
(Gaspard N. and Pierre V., 2009)

40. 40
Fig. 1. Diagram showing the major divisions of the vertebrate nervous
system.. (Human Anatomy & Physiology, 2007)

41. hESC neural differentiation medium
41
(1) rosette formation
Chemical Final Concentration
DMEM/F12 (Gibco, Rockville) Basic medium
β-Mercaptoethanol (Invitrogen) 1:100
MEM-NEAA (Inventrogen) 1:100
Heperin (Sigma) 2 μg/ml
FGF2 (R&D Systems) 20 ng/mL
(Johnson et al., 2007)
(Johnson et al., 2007)

42. hESC neural differentiation medium
42
(2) differentiating into primitive neuroepithelial (NE) medium
(Johnson et al., 2007)
(Hu et al., 2009)
Chemical Final Concentration
DMEM/F12 (Gibco, Rockville) Basic medium
MEM-NEAA (Inventrogen) 0.1 mM
β-Mercaptoethanol (Sigma) 0.1 mM
Heperin (Sigma) 2 μg/ml
Acrobic acid (Sigma, cat. No. B1934) 200μM
BDNF (PepeoTech, cat. No. 450-02) 10 ng/mL
GDNF (PepeoTech, cat. No. 450-10) 1:10000
IGF-1 (PepeoTech, cat. No. 100-11) 1:10000
Cyclic AMP (PepeoTech, cat. No. D-0260) 1 μM