This presentation summarizes some of the most popular neural differentiation protocols. It also contains some of the most recent developments in these protocols including small molecule based methods.

1. Neural Differentiation
Amir Motmaen
Summer 2016

2. Overview
• NSC derivation
• Neuronal differentiation
• Astrocyte differentiation
• Oligodendrocyte differentiation

3. NSC Derivation

4. Rosette/EB-based
Protocols

5. NSC derivation from rosette
formation of embryoid bodies
• aggregates of PSCs in suspension
• EB attachment to matrix
• generation of neural rosettes
• exp. of early neuroectodermal markers
• NSCs from rosettes using conventional
morphogens

6. Retinoic acid protocol
• RA posteriorizes CNS tissue in dev. also plays a role in adult
hippocampal neurogenesis
• promotes diff. of pluripotential teratocarcinoma cells to neural
progenitors and neurons
• embryoid bodies (4-/4+) then adhesion to substrate for 7d
• 40% neuron-like cells
• restricted diff. potential
• numerous other cell type in final product
• length of culture time up to 2w
Bain et al., 1995; Bibel et al., 2004

7. MEDII conditioned medium
induction
• CM of human hepatocellular carcinoma cell line
Hep-G2 promotes homogenous PEL(primitive
ectoderm-like) from ESC
• EB with no OVM
• essentially pure neuroectoderm-like epithelial layer
• no regional identity
Rathjen et al., 2002

8. EB selection in defined
medium
• EB intermediated in which all 3 primary germ layers arise followed
by neural lineage-specific selection
• EB to substrate, minimal serum-free ITSFn medium (insulin,
transferrin, selenium, fibronectin)
• other cells die during several days of culture
• subsequent plating on laminin + FGF2 for NSC proliferation
• relative purity of final product (80-95%)
• large numbers of NSC upon FGF2 addition
• extended time & variability in quality which can be contaminated
Okabe et al. 1996

9. Derivation Of NSCs By
Direct Differentiation

10. Culture at very low densities
in defined medium
• 1-20 cells/well, feeder free, serum-free defined medium
• primitive NSCs as intermediate cell type
• ESCs to LIF-dep. primitive neurospheres (Nes+,Oct4+)
• maturation to LIF-ind. FGF/EGF-dep. neurospheres (Nes+,Oct4- definitive NSCs)
• inhibited by BMP4 & promoted by noggin
• forebrain & hindbrain markers
• 0.2% ESC to sphere-forming colonies
• inefficient for NSC production but used for assessment of putative survival
& proliferation factors
Tropepe et al., 2001; Smukler et al., 2006

11. Feeder-free ESCs cultured
at moderate densities
• better using ESCs adapted to feeder-free
• 104/cm2 on gelatin-coated dishes, -LIF, serum-free defined medium
• NSC production in part by selection
• rapid direct emergence of NSCs (4-6d)
• pure isolation using FACS, purity improves by trypsin-based
passage
• efficient for NSC production
• presence of other cell types
Ying et al., 2003

12. Stromal cell co-culture
• stromal cell can induce direct diff. of NSCs from ESCs
• ESCs plated at clonal densities on stromal cell lines
• NSC diff. apparent at d6
• cells plated at low density to minimize cell-cell interactions
& serum replacement instead of serum
• relatively pure multipotent neural derivatives that can be
induced to sub-types with high efficiency
• absence of EB reduces time to a few days
Barberi et al., 2003; Kawasaki et al., 2000

13. combines use of an embryoid body in-
termediate, in which cells of all three
primary germ layers arise, followed by
a neural lineage-specific selection step
(Okabe et al., 1996; Guan et al., 2001;
Wobus et al., 2001). This approach be-
gins with early stage embryoid bodies.
These are then plated on adhesive
substrates in a minimal serum-free
medium, ITSFn, which contains insu-
lin, transferrin, selenium, and fi-
bronectin (Table 1; Fig. 1, protocol 3).
Fig. 1. Protocols for producing embryonic stem cell (ESC) -derived neural stem cells (NSCs). Protocols 1–3 include an embryoid body intermediate,
whereas 4–6 are embryoid body-independent. Protocols 1–5 begin with feeder-dependent ESCs, whereas protocol 6 is most efficient when applied
to feeder-free lines.
Cai et al., 2007
combines use of an embryoid
termediate, in which cells of a
primary germ layers arise, foll
a neural lineage-specific select
(Okabe et al., 1996; Guan et a
Wobus et al., 2001). This appr
gins with early stage embryoid
These are then plated on a
substrates in a minimal ser
medium, ITSFn, which contai
lin, transferrin, selenium,
bronectin (Table 1; Fig. 1, pro
During several days of cultu
neurectoderm cells, including
Fig. 1. Protocols for producing embryonic stem cell (ESC) -derived neural stem cells (NSCs). Protocols 1–3 include an embryoid body inte
whereas 4–6 are embryoid body-independent. Protocols 1–5 begin with feeder-dependent ESCs, whereas protocol 6 is most efficient whe
to feeder-free lines.

14. TABLE 1. Comparison of Protocols Using ESCs to Derive NSCsa
Protocols
1. RA induction (4Ϫ
/4ϩ)
2. MEDII
CM induction 3. Serum-free selection 4. Stromal co-culture
5. Low density clonal
neurosphere 6. Monolayer Serum-free
Mouse ESC
lines used
D3, CCE E14, D3 J1, CJ7, D3, R1 ESC: CJ7, AB2.2,E14,ESB5
ntES:C4, C15,C16,CN1,2,
CT2
R1 46C (E14 derived) 15
clones of ESC
Coculture,
CM, or other
factors
None HepG2
conditioned
medium
None Stromal cell (MS5,S17,PA6
etc)
LIF none
Initial plating
density
Not quantified
Ͼ1ϫ105
cells/ml
1ϫ105
cells/
ml
2-2.5ϫ104
cells/cm2
50 cells/cm2
1-20 cells/microwell 0.5-1.5x104
cells/cm2
Culture type Suspension Suspension Suspension ϩ adherent Adherent Suspension Adherent
EB formation Yes Modified Yes No No No
Serum or
serum
replacement
10% FBSϩ 10%
newborn calf serum
10% FBS 10% FBS for EB then no
serum
15% serum replacement No serum No serum
Days to reach
NSC peak
8 7 10-12 6 3 (4 hrs in PBS) 5
% NSC at
peak
39% neuron-like
Cellsb
Nearly 100%
95.7%
NCAMϩ
Ͼ80% High, not quantified 100% 75%
NSC marker ␤III tubulin Sox1, Sox2,
nestin
nestin nestin, NCAM, Musashi nestin Sox1, nestin
NSC Regional
identity
NA Otx1 (fore-
and
midbrain)
En1, En2
(midbrain)
Otx1 (fore- and midbrain)
En1 (midbrain)
No specific regional identity Emx2 (forebrain) HoxB1
(hindbrain)
NA
Differentiation
potential of
derived NSC
NA Neurons, glia
(Ͼ95%),
neural crest
Neurons (Map2) Astrocytes
(GFAP) Oligodentrocytes
(O4)
Neurons (dopaminergic,
serotonin, GABAergic and
motor neurons with high
efficiency), glia
Neurons (Map2) Astrocytes
(GFAP) Oligodendrocytes
(O4)
Neurons (GABA, TH)
Astrocytes (GFAP)
Oligodendrocytes
(CNPase)
Other lineages Many other lineages
present
None Non-neural lineages
selected against in serum-
free medium
None primitive endoderm present
(GATA4), no mesoderm or
definitive endoderm
Non-neural cell types and
Oct4ϩ cells present
Key
references:
Bain et al., 1995
Bibel et al., 2004
Rathjen et
al., 2002
Okabe et al. 1996 Barberi et al., 2003
Kawasaki et al., 2000
Tropepe et al., 2001
Smukler et al., 2006
Ying et al., 2003
a
ESCs, embryonic stem cells; NSCs, neural stem cells; RA, retinoic acid; CM, conditioned medium; NA, not applicable; GFAP, glial fibrillary acidic protein; FBS, fetal bovine serum;
NCAM, nerve cell adhesion molecule; En1-2, Engrailed 1-2; MAP2, microtubule associated protein 2; GABA, ␥-aminobutyric acid.
b
Stem cells or progenitors not assayed.
Cai et al., 2007

15. Rosette/EB free dual SMAD inhibition
• Noggin (BMP inh.) & SB431542 (Lefty/Activin/TGF-β inh.)
• completed in 11 d
• exp. of Sox1 preceding Pax6 despite previous reports
• yields an early Pax6+ neuroepithelial population capable of rosette
formation
• A/P & D/V & subtype dependent on early exposure to morphogenic
factors
• first highly efficient report bypassing EB
Chambers et al., 2009
erica,Inc.Allrightsreserved.
g h
a
e
b c
f
k l
d
Day: 1 5 9
KSR
SB431542
12 19
m
Noggin
11
TH neuron:
Motoneuron:
BAGTCSHH BASF
BASR Passage
N2
Nestin, PAX6 PLZF, PAX6 ZO1, PAX6 pHH3, KI67
OTX2, PAX6 FOXG1, PAX6 AP2, PAX6 HNK1, PAX6
PAX7, PAX6 p75, PAX6 Pigment Melanosome (HMB45)
TH, TUJ1TH, TUJ1 TH, TUJ1 ISL1 HB9
i j
Figure 2 Neuralization of hES c
SMAD inhibition permits a pre-r
stem cell with dopaminergic and
potential. (a–c) The PAX6+ neur
expressed rosette markers (red)
(b), ZO1 (c). (d) Rosettes are fo
tissue is passaged to conditions
rosettes (BASF) confirmed by KI
luminal phospho-histone H3 (re
evidence of interkinetic nuclear
(e,f) In the absence of factors th
neuronal specificity, the PAX6+
(green) expressed OTX2 (e) and
indicating that the tissue defaul
specification. (g–j) Neural crest
identified on the periphery of th
(green) based on AP2 (g), HNK1
and p75 expression (j) (red). (k,
the neural crest cells gave rise t
(k) that expressed HMB45 (l; gr
melanosome synthesis. (m) Dop
neuronal patterning was initiated
addition of super sonic on days
by the addition of BDNF, ascorb
hedgehog and FGF8 on days 9–
cells were matured on days 12–
ascorbic acid, GDNF, TGFb3 and
Motoneuronal patterning was ini
with the addition of BDNF, asco
hedgehog, and retinoic acid. Ce
on day 11. (n,o) Without passag
could be observed by day 19. (p
en bloc on day 12, more mature
TH+ cells were observed. (q,r) F
induction, nuclear expression of
markers ISL1 (q) and HB9 (r) w
L

16. SB431542
TGF-β
CNS (PAX6)High density
Trophectoderm
SB431542
Activin
and
Nodal
Noggin
BM
P
M
esendoderm
Ectoderm
Noggin
BM
P
Passage
Rosette NSC
Patterning
Motoneurons or
dopaminergic neurons
Patterning
Days 1–6
Days 6–19
OCT4 PAX6
SB431542
Trophectoderm
Noggin SB431542
TGF-β
Activin
and
NodalBM
P
Low density CNS (PAX6) and PNS (p75, HNK-1)
M
esendoderm
Ectoderm
Noggin
BM
P
FACS
(PNS)
Neural crest SC
(p75, HNK-1)
Patterning
Patterning
Neural crest
progeny
Passage(PNS?)
Early melanocytes
(HMB45)
a
b
SOX1 (RNA)
OCT4 PAX6
p75 (RNA)
Days 1–6
y
by
ls,
e
NS
n
in a
est
t
o
on
ys,
t
d
VOLUME 27 NUMBER 3 MARCH 2009 NATURE BIOTECHNOLOGY
Chambers et al., 2009

17. Neuronal Differentiation

18. Neural tube & Neural crest
lineages
• based on the idea of cells from border of neural plate
• small molecules only, for HTS
• smNPCs using EB by DM + SB + CHIR + PMA from hESs
• altering CHIR & PMA conc.: together for expansion,
CHIR: neural crest, PMA: ventral neural tube lineages
• permissive diff. to all 3 basic lineages
• direct diff. to PNS neurons, mesenchymal, mDANs, MNs
Reinhardt et al., 2013

19. TH+/CASP3- (empty arrowhead) and TH+/CASP3+ neurons (arrowhead). (B) When normalized to the average number of apoptotic cells detected in
the wild-type cultures, 6-OHDA and rotenone lead to a higher cell death, with an even higher increase in cells carrying LRRK2 G2019S. Error bar
represent the variation from duplicate wells. (C) When normalizing each concentration to the average apoptosis in TH+ neurons from healthy
controls, an increase of 46% can be observed in LRRK2 G2019S over wild type cultures in all stressor concentrations used. Error bars represent S.D. ***
indicates p,0.001, according to Student’s t-test. See also Figure S11 for primary, unnormalized data.
doi:10.1371/journal.pone.0059252.g007
Figure 8. Summary of smNPCs. Diagram illustrating the conditions used to derive, propagate, and differentiate smNPCs. CHIR = 99021
DM = dorsomorphin, FCS = fetal calf serum, PMA = purmorphamine, RA = all-trans retinoic acid, and SB = SB43152.
doi:10.1371/journal.pone.0059252.g008
PLOS ONE | www.plosone.org 12 March 2013 | Volume 8 | Issue 3 | e59252
Reinhardt et al., 2013

20. Table 1. Summary of the markers used in this study as well as the characteristics of NSCs, lt-hESNSCS, R-NCs, pNSCs, and smNPCs.
Cultured Cell Type
Markers NSCs lt-hESNSCs pNSCs R-NCs smNPCs
Origin Not applicable Fetal/adult brain hPSCs hPSCs hPSCs hPSCs
Immortal self-renewal? Not applicable Yes Yes Yes No (very limited
expansion)
Yes
Self-renewal with only small molecules? Not applicable No No No No Yes
Differentiation
potential
Neural crest PAX3, MSX1, SOX9, SLUG, PAX7,
TFAP2A
No Not tested No Yes Yes
Peripheral neurons (neural crest-derived) PERIPHERIN, BRN3A, TFAP2A No Not tested No Yes Yes
Mesenchymal cells (neural crest-derived
lineage)
NESTIN, VIMENTIN, CD9 No Not tested No Not tested Yes
Osteoblasts (mesenchymal cell-derived) SMA No Not tested No Not tested Yes
Osteocytes (mesenchymal cell-derived) OSTEOCALCIN, AP No Not tested No Not tested Yes
Adipocytes (mesenchymal cell-derived) FABP4 No Not tested No Not tested Yes
Neural rosettes (neural tube lineage) ZO1, DACH1, PLZF, LMO3, NR2F1,
PLAGL1, EVI1, LIX1
No Not tested Yes Yes
Neural progenitors (neural tube lineage) PAX6, SOX1, SOX2, NESTIN Yes Yes Yes Yes Yes
Ventral neural tube (neural tube-derived
lineage)
NKX6.1, NKX2.1, NKX2.2,
OLIG2, FOXA2
Mixture Yes Yes Yes Yes
mDANs (ventral neural tube-derived lineage) TH, FOXA2, EN1, LMX1A, LMX1B,
NURR1, AADC
No/very few Yes (30%) Yes (69–80%) Yes Yes (70%)
MNs (ventral neural tube-derived lineage) ISL1, HB9, CHAT, SMI32 No/very few Yes (15%) Yes (71%) Yes (25%) Yes (50%)
Neurons TUJ1, MAP2, NeuN, DCX,
SYNAPTOPHYSIN
Yes Yes Yes Yes Yes
Astrocytes GFAP, S100-b Yes Yes Yes Yes Yes
Oligodendrocytes O4, OLIG2 Yes Yes No Yes Yes
Differentiation efficiencies are reported in % of neurons and not of total cells.
doi:10.1371/journal.pone.0059252.t001
Reinhardt et al., 2013

21. Direct diff. of cortical interneurons
from hESCs
• Robust FOXG1/PAX6 by NSB
• LDN193189 (ALK2/3 inh.) instead of Noggin: better PAX6, lower
FOXG1
• two inhibitors of canonical Wnt: recombinant DKK1 or XAV939
(tankyrase inh.) enhanced FOXG1 in LSB-treated cultures
• first study to achieve robust forebrain lineage using 3 small molecules
• SHH : 1µM PMA + 5µM SHH
Stem Cell
C-Derived Cortical Interneurons
Maroof et al., 2013
Cell Stem Cell
hESC-Derived Cortical Interneurons
Figure 1. Wnt Inhibition and Activation of SHH Signaling Yields Highly Efficient Derivation of Forebrain Fates and N
(A) Schematic of the differentiation protocol in the dual-SMAD inhibition paradigm for generating anterior neural progenitors.
Cell Stem Cell
hESC-Derived Cortical Interneurons

22. Timing of SHH exposure for specific
ventral precursors
• 2-18 d: suppression of FOXG1 induction, hypothalamic
fate (RAX) without FGF8, dopaminergic (TH)
• 6-18 d & 10-18 d: both FOXG1+ but differences in OLIG2
thus different NKX2.1 as in natural development,
cholinergic & cortical interneurons (GABAergic)
respectively
Cell Stem Cell
hESC-Derived Cortical Interneurons
Maroof et al., 2013
Figure 2. Timing of SHH Exposure Determines the Regional Identity of NKX2.1::GFP-Expressing
(A) Model of human prosencephalon (sagittal view at CS14) with expression of forebrain patterning markers ba
diencephalon; Tien., telencephalon.
(B–E) Coronal (oblique) hemisection of the human prosencephalon at CS15 demonstrates expression of NK
expressed in various regions throughout the ventral prosencephalon, whereas PAX6 is restricted to the dorsa
preoptic area; HYPO, hypothalamus. The scale bar in (B) represents 200 mm. The expression of these pro
eminence (GE) (C), where OLIG2 and NKX2.1 are coexpressed.
Figure 1. Wnt Inhibition and Activation of SHH Signaling Yields Highly Efficient Derivation of Forebrain Fates and NKX2.1 Induction
(A) Schematic of the differentiation protocol in the dual-SMAD inhibition paradigm for generating anterior neural progenitors.
(B–E) When either DKK1 or XAV939, both Wnt-signaling antagonists, was added to the dual-SMAD inhibition protocol (DLSB or XLSB), there was a sig
increase in the percentage of FOXG1+ cells (B) without a loss of PAX6 expression (C): **p < 0.01; ***p < 0.001; n.s., not significant; using ANOVA follo
Scheffe test. (D) Representative immunofluorescent image for FOXG1 (red) and PAX6 (green) expression at day 10 following XLSB treatment. Single-c
Cell Stem Cell
hESC-Derived Cortical Interneurons
C
hESC-Derived Cortic

23. hPSC-derived MGE-like progenitors
• telencephalic MGE-like identity
• exhibited VZ & SVZ radial glial-like stem cell behavior
• diff. into GABAergic neurons
• protracted maturation times consistent with dev.
Cell Stem Cell
MGE GABAergic Interneuron Maturation from hPSCs
Nicholas et al., 2013

24. (No´ brega-Pereira
finding suggests
of the neurons de
ments are inde
interneurons. Thi
tiated by an impo
tion experiments
and colleagues
GABAergic neuro
migrate toward
planted into the
All together, the
that bona fide c
neurons can be
pluripotent stem
worth emphasiz
are heterogeneo
classes of GABA
striatal interneur
will be necessary
ential derivation
fated to produce
One of the mo
the studies by M
Nicholas et al. (2
that GABAergic
from human pluri
an extremely exte
which somehow m
opment of thes
In vitro, the proto
and colleagues a
Figure 1. Normal Development Guides the Derivation of Human GABAergic Interneurons
In Vitro
Marin, 2013

25. Regionally specified progenitors & functional
neurons from hESCs under defined conditions
• telencephalic identity & mature glutamatergic neurons using dual inh.
• caudalization by Wnt using CT99021(CHIR) thus all progenitors from telencephalon
to posterior hindbrain
• dorsoventral identity at the same time: withdrawal of NSB: +SHH for ventral & -SHH
for dorsal
• mesDA progenitors in VM conditions (0.7-0.8µM CT + SHH): DAN in vitro & in vivo
BMP signaling in
markers FOXA1, N
markers SIM1, NK
presence of SHH (
for expression of fl
concentrations of
showed a tendenc
SHH, PAX6 as we
WNT1 were expr
markers was partic
from the medium o
scription factor LM
withdrawal, reflect
which is present in
oping human midb
hindbrain markers
affected by pattern
Transplanted Ne
and Caudal Fates
Tumor-free Graft
We next transplan
identity after 10 or
adult rats. Groups
hESCs correspon
fates: rostral d10
(SHH + 0.7 mM CT
d10 (SHH + 2 mM C
observed that all of
transplants and no
features such as
the transplanted c
obvious contamina
ified cells tended t
VM-specified cells
Kirkeby et al., 2012

26. FGF8 for DA neurons
• FGF8 traditionally as a factor for patterning the
midbrain (isthmus) identity
• FGF8 promotes the DA diff. of midbrain progenitors
but not the more caudal progenitors1660 Specification of Midbrain DA Neurons
Xi et al., 2012

27. Improved protocol for PD based on
diff. efficiency & safety
• method A most efficient, CHIR better than Wnt1
• high CHIR in C&D lowers efficiency best in 1µM
comparable with A&B1550 Differentiation and Sorting of DA Neurons
Sundberg et al., 2013

28. A practical approach for derivation of
DAN for clinical use
• neural inducers: SB218078 (stauprimide homolog) &
DMH-1 (DM homologe)
• GS (guggulsterone, a naturally occurring steroid) for DAN
diff. induction after priming for DANs with PMA & FGF8
• GS used instead of: mix of BDNF, GDNF, TGFβ3, DAPT,
dbcAMP
• 5-fold more DA in GS-treated cells vs control (DMSO
instead of GS) also secreting 3-fold more dopamine in vitro
Gonzalez et al., 2013

29. Derivation of motor neurons
• SM induction of neuralization, caudalization & ventralization
• intermediary EB formation
• SAG (Smo agonist) & PUR (Purmorphamine)
• S+P protocol: inc. FOXP1, dec. LHX3, RALDH2+ thus LMC
identity; elevated HOXA5+ & HOXC6+ also HOXC8 but no
HOXC9 or HOXC10 so rostral brachial identity
Amoroso et al., 2013
PUR 1 (RA
GFPcells(%ofDAPI)
+
HAG1 uM SHH SAG0.5
uM(RA
Control
SHH
200
ng/m
L
SAG
1
Mµ
PUR
1
Mµ
35
30
25
20
15
10
5
0
35
30
25
20
15
10
5
0
GFPcells(%ofDAPI)
+
RA
0.1
Mµ
RA
1
Mµ
GFPcells(%ofDAPI)
+
35
30
25
20
15
10
5
0Control
SAG+PUR
B C
*
SAG
1
Mµ
PUR
1
Mµ
ES/iPS Embryoid Bodies
S+P
SHH
Day 5 Day 7 Day 17 Day 21 Day 31
RA
SB435142
LDN193189
NTFs
Y27632
bFGF
CondiƟon 1:
CondiƟon 2:
Day5 Day14 Day21 Day31
Day 0
HAG
1
Mµ
tem Cell-Derived Limb-Innervating MNs J. Neurosci., January 9, 2013 • 33(2):574–586 • 577
LHX3ISL1FOXP1
Limb Non-Limb
FOXP1LHX3
C
FoldchangeS+P/SHH(Log)
LHX3
RALDH2
FOXP1
.5
1
2
4
axial
limb
*
*
MMC
LMC
BA
Limb Level
MMC:LHX3
LMC: RALDH2, FOXP1
+
++
2
582 • J. Neurosci., January 9, 2013 • 33(2):574–586 Amoroso, Croft et al. • Human Stem Cell-Derived Limb-Innervating MNs
A
LMC : RALDH2, FOXP1
LMC : RALDH2, FOXP1, LHX1
m
l
+ +
+ + +
B
Limb
LMC
LMCm
LMC Divisional IdenƟty
l
B C
FoldchangeoverES
0
5
10
15
20
25
30
35
HOXA5 HOXC6 HOXC8 HOXD9
S+P: GFP+
SHH: GFP+
ES control
Amoroso, Croft et al. • Human Stem Cell-Derived Limb-Innervating MNs
A
LMC : RALDH2, FOXP1
LMC : RALDH2, FOXP1, LHX1
m
l
+ +
+ + +
B
Limb
LMC
LMCm
LMC Divisional IdenƟty
l
B C
FoldchangeoverES
0
5
10
15
20
25
30
35
HOXA5 HOXC6 HOXC8 HOXD9
S+P: GFP+
SHH: GFP+
ES control
Amoroso, Croft et al. • Human Stem Cell-Derived Limb-Innervating MNs

30. HTS suitable method
• fast, reproducible and reliable similar to Amoroso
but can be expanded for HTS
• intermediate EB, DM & SB, RA for induction
• BDNF, GDNF, CNTF or Kenpaullone for maturation
• Kenpaullone (GSK-3 inh.) better survival/maturation
factor
Yang et al., 2013

31. Astrocyte Differentiation

32. Transplantable astroglia from hPSC
• generation of large quantities but in an extended time
similar to dev. (180d)
• region specification same as in neuronal specification
Krencik et al., 2011
A RT I C L E S
b S100 /Ho GF
180days
a
hPSC NE Progenitors Astroglia
0 days 10 21 180
Neural induction Patterning Expansion
(EGF + FGF2)
lll-tubulin
NFIA, S100
CD44, GFAP
(FGF8, RA, or SHH)
906030
d GFAP/Aldh1L1/Ho GFAP/NG2/Ho
ry
e RA FGF8 g NFIAPAX6Ho
c
Percent
S100
+
/totalcells
100
*
80
60
40
20
30 60 90 120Days
RA FGF

33. Astrocytes from hPSCs using a
defined system
• chemically defined and xeno-free
• CD44+ Intermediate Stage that can be Cryopreserved
• BMP & CNTF can directly differentiate this precursor
• Neuregulin also promote astrocyte diff.
• NFIA (nuclear factor 1A) & HES (hairy and enhancer of split) pathways are
potential regulators of cell fate
• 4-6 w, cryopreservation at several stages, defined medium
Shaltouki et al., 2013

34. Oligodendrocyte Differentiation

35. Oligodendrocytes from
hPSC
• caudalization & multi potency inh. by RA
• novel GRM (glial restriction media)
• GRM: insulin, triiodothyroidin, FGF, EGF
Nistor et al., 2005

36. Zeno-Free culturing protocol for oligodendrocyte
precursor cell production
• StemPro®
neural stem cell xeno-free medium
• supplement together with human recombinant growth factors
SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition
to RA, T3, human laminin and ascorbic acid.
ReseaRch aRticle Sundberg, Hyysalo, Skottman et al.
Neural induction of hESCs
0 weeks
Adherent
for 2 weeks
Adherent
for 1 week
Adherent
for 1 week
FACS
ICC
FACS
ICC
FACS
ICC
Differential components
RA
SHH
EGF
bFGF
T3
AA
± CNTF
Laminin
PDGF-AA
IGF-1
qRT-PCRqRT-PCRqRT-PCRqRT-PCRqRT-PCR
4 weeks 5 weeks 7 weeks 9 weeks
OPC production OPC differentiation
Figure 1. Human embryonic stem cell-derived oligodendrocyte precursor cell differentiationSundberg et al., 2011