Table 2.
Differentiation methods to produce medium spiny neurons, cortical neurons, astrocytes, and oligodendrocytes from ESC and iPSCs.
| Starting Cell Type |
Differentiated Cell Type |
Differentiation Method | Markers | Efficiency | Reference |
|---|---|---|---|---|---|
| ESC | MSN | d0-12: co-culture hESC with MS cells in DMEM-F12/KSR; d12–21: culture in DMEM-F12/N2; d21: isolate neural rosettes and plate on poly-ornithine/laminin surface; d21–46: culture in DMEM-F12/N2 with BDNF, SHH, DKK1; d46–62: differentiate into neuron in DMEM-F12/N2 with BDNF, db-cAMP, VPA |
DARPP32 GABA calretinin calbindin |
53% DARPP32+ in MAP2+ cells |
78 |
| ESC | MSN | d0-6: EB in suspension; d6: attach on plastic surface; d6–10: culture in DMEM-F12/N2; d10–30: induce ventral telencephalic progenitor in DMEM-F12/N2 with SHH, DKK1; d30–42: differentiate into neuron in Neurobasal/N2/B27/BDNF/GDNF/IGF1 |
DARPP32 GAD65/67 GABA |
not described |
79 |
| ESC | MSN | d0-12: primitive neuroepithelia induced in DMEM-F12/N2; d12–26: SHH or purmorphamine added in DMEM-F12/N2; d26–33 : Neurobasal/VPA; d33-: Neurobasal/BDNF/GDNF/IGF/cAMP |
DARPP32 GAD65/67 GABA Meis2 |
89.7% DARPP32+ in GABA neurons |
85 |
| ESC and iPSC | MSN | d0-3: PSCs expanded in MEF conditioned medium with Y27632; d3–15: KSR medium with dorsomorphin and SB431542; d8–29: SHH and DKK1 added; d29-d80 : terminal differentiation in N2/B27 medium with BDNF |
DARPP32 GAD65/67 GABA calbindin CTIP2 |
20% DARPP32+ |
80 |
|
Immortalized NPC line |
MSN | Differentiation in DMEM-F12/N2/B27/lnsulin; d0-d11: BDNF (30ng/ml)/SHH/DKK1; d0-d1: additional Y-27632 added; d11- 60: BDNF (50ng/ml) |
DARPP32 GABA Calbindin |
21% DARPP32+, 96% GABA+, 84% Calbindin+ in MAP2+ neurons |
82 |
| ESC and iPSC | MSN | d0-d5: 80% confluent PSCs induced in DMEM- F12/Neurobasal (2:1) supplemented with N2/B27/SB431542/LDN-193189; d5-d9: SB431542 removed; d9–28: LDN-193189 replaced by activin A; d28–36: BDNF/GDNF added |
DARPP32 CTIP2 Calbindin |
20–50% DARPP32+ |
83 |
| ESC and iPSC | Cortical Neuron | Differentiation in 3N medium (DMEM-F12/Neurobasal, 1;1, N2/B27/insulin/vitamin A). d0-d11: 95% confluent monolayer PSCs on Matrigel differentiated in 3N with retinoids, SB431542 and dorsomorphin or noggin; d11–13: replatedto poly-ornithine/laminin in 3N with bFGF; d13–100: maintained in3N |
Pax6 01×1/2 Emx1 Foxgl Tbr2 |
>95% Pax6+, Otx1/2+by d15 |
98 |
| ESC and iPSC | Cortical Neuron | d0-16: monolayer PSCs on Matrigel differentiated in DMEM- F12/N2/B27/BSA/Glutamax/noggin; d16–24: same medium with noggin removed; d24: replate cells to poly-lysine/laminin in same medium with noggin removed and Y27632 added; every 5–7d, half medium changed with Neurobasal/B27/glutamine |
Pax6 Otx1/2 Emx1/2 Foxgl Tbr1/2 vGlut1/2 |
Mostly Pax6+, Oxt1/2+ d10–19, 60% vGlut1 + d44 |
99 |
| NSC | Astrocyte | NSCs on poly-ornithine/laminin or Geltrex were induced for astrocyte differentiation with StemPro/activin A/Heregulin 1β/IGF1 |
GFAP S100β |
62% GFAP+ |
108 |
| ESC and iPSC | Astrocyte | d0-6: EB in suspension; d6: EB plated on Matrigel in Neurobasal/N2; d6–9: bFGF/EGF added; d9–12: bFGF/EGF/CNTF added; d12–15: bFGF/CNTF added; d15-: CNTF alone; |
GFAP S100β GLT-1 |
78% GFAP+ at 5weeks |
109 |
| iPSC | Astrocyte | iPSCs were induced with dorsomorphin/SB431542/NAC for 5–7d before enzymatic dissociation to form neurospheres; retinoic acid added to neurospheres for 7–12d; neurospheres mechanically chopped and replated in NSCR (AdDMEM- F12/N2/B27/NEAA/Glutamax) with EGF/LIF for 2–4w; propagated in NSCR with EGF/bFGF; dispase dissociation and CNTF induced differentiation |
GFAP sioop Vimentin NF1A |
>90% GFAP+, S100β+ |
110 |
| ESC and iPSC | Oligodendrocyte progenitor cells (OPCs), Oligodendrocytes (OL) |
d0-d8: neural induction using dual SMAD inhibition and 100 nM all-trans RA in adherent conditions; d8: added SAG to medium; d12: lift cells for sphere formation in medium with RA and SAG; d30: plate spheres on poly-L-ornithine/laminin dishes in PDGF medium for OPC formation; d75+: glial medium for oligodendrocyte maturation |
OPC: NKX2.2 SOX 10 OLIG2 OL: O4 MBP |
40–70% O4+ |
115 |
| ESC and iPSC | Oligodendrocyte progenitor cells (OPCs), Oligodendrocytes (OL) |
d0-d5: EB formation in ESC medium; d5-d19: neuroepithelial generation in NIM, FGF2; d19-d29: pre-OPC generation in neural induction medium (NIM), RA, B27, purmorphamine; d29-d40: pre-OPC expansion in NIM, bGFG, B27, purmorphamine; d40-d160: OPC generation in glial induction medium (GIM), T3, NT3, IGF, PDGF-AA, purmomorphamine; d160-d180: terminal differentiation in reduced GIM |
OPC: OLIG2 NKX2.2 SOX 10 PDGFRa 0L04 MBP |
OPC: >45% ESC derived >70% iPSC derived |
116 |