Table 3.
Current protocols for astroglial differentiation of human PSCs.
| Reference | Cell source | Method of Differentiation | Key players | Research Outcome | Early Markers | Mature/Late Markers |
|---|---|---|---|---|---|---|
| Zhang et al., 2001 | hESCs | EB | FGF-2: 20 ng/ml cAMP: 100 ng/ml BDNF: 10 ng/ml PDGF-A: 2 ng/ml | GFAP+ both in vitro and in vivo | Nestin, Musashi-1, PSA-NCAM | NF200, GFAP |
| Carpenter et al., 2001 | hESCs | EB | RA: 10 μM hEGF: 10 ng/ml hbFGF: 10 ng/ml hPDGF-AA: 1 ng/ml hIGF-1: 1 ng/ml hNT-3: 10 ng/ml hBDNF: 10 ng/ml | – | Nestin, PSA-NCAM, A2B5, MAP-2, Synaptophysin | |
| mESCs | NS | – | Vimentin, NF1A, GLAST, ALDH1L1, GLT-1 | GFAP, AQP4, S100β | ||
| Tabar et al., 2005 | hESCs | ML | FGF2: 20 ng/ml EGF: 20 ng/ml Noggin: 500 ng/ml SB 431542 | At fourth week, 2% expressed astrocyte marker | Nestin, calretinin, DLX2, NCAM, A2B5 | β-III -Tubulin, EGFAP |
| Itsykson et al., 2005 | hESCs | EB | Glial fate observed at 25th week | GABA, glutamate, serotonin, tyrosine hydroxylase, O4 | GFAP, β-III-Tubulin, | |
| Johnson et al., 2007 | hESCs | NS | Heparin: 2 μg/ml FGF2: 20 ng/ml BDNF: 10 ng/ml GDNF: 10 ng/ml cAMP: 1 μM Ascorbic acid: 200 μM | By nineth week astrocyte appeared in the neural network | For synaptic analysis MAP2, Synapsin-1, β-III-Tubulin, | GFAP, S100β |
| Hu et al., 2010 | hESCs and iPSC | EB | Glial: RA: 100 nM SHH: 100 ng/ml cAMP: 1 μM Oligodendrocytes: PDGF-AA: 60 ng/ml Neurotrophin 3: 10 ng/ml IGF1: 10 ng/ml | GFAP+ cells after 3 months and excitatory postsynaptic currents were observed in >8 weeks culture (but efficiency unknown) | β-III -Tubulin, S100β | GFAP |
| Krencik., 2011, 2012 | hESCs and iPSC | EB | RA: 0.5 μm FGF8: 50 ng/ml SHH: 500 ng/ml EGF and FGF2: 10 ng/ml CNTF: 10 ng/ml LIF: 10 ng/ml | Uniform populations of immature astrocytes (>90% S100β+ and GFAP+). | For synaptic analysis MAP2, Synapsin-1, β-III-Tubulin, | GFAP, S100β |
| Hayashi et al., 2011 | rat iPSC | NS | FGF-2: 20 ng/ml FBS: 10% | NSC differentiated exclusively into astrocytes when FGF-2 was removed from neurobasal medium | Nestin, β-III-Tubulin | GFAP, S100β |
| Emdad et al., 2012 | hESC and hiPSC | EB | SB43152: 10 μM Noggin: 500 ng/ml | 55–70% of GFAP+ cells at week 5 | Nestin, GLT-1, A2B5, β-III-Tubulin | GFAP, GLAST, aquaporin-4 |
| Juopperi et al., 2012 | hiPSC | EB | bFGF: 20 ng/ml | S100β and GFAP+ cells after 2–3 months (efficiency unknown) | Nestin, β-III-Tubulin, MAP2ab, doublecortin (DCX) | GFAP, S100β |
| Lafaille et al., 2012 | hiPSC | EB | EGF/FGF2: 20 ng/ml SonicC25II: 125 ng/ml FGF8: 100 ng/ml BDNF: 20 ng/ml Ascorbic acid: 0.2 mM | 90% GFAP+ cells after 60–90 days | Nestin, β-III-Tubulin | GFAP |
| Serio et al., 2013 | hiPSC | EZS/ NS | EGF/FGF2: 20 ng/ml CNTF: 10 μg/ml | After ∼8 weeks, 90% cells positive for GFAP | Vimentin, nuclear factor 1A | GFAP, S100β |
| Shaltouki et al., 2013 | hESC and iPSC | EB | bFGF: 20 ng/ml CNTF: 5 ng/ml BMP: 10 ng/ml bFGF: 8 ng/ml Activin A: 10 ng/ml Heregulin 1β: 10 ng/ml IGFI: 200 ng/ml | 60–80% of GFAP positive cells after 5 weeks (starting from NSC). | β-III -Tubulin | GFAP, S100β |
| Roybon et al., 2013 | mESC; hESC hiPSC | ML | LDN193189: 0.2 μM SB431542: 10 μM Ascorbic acid: 0.4 μg/ml RA: 1 μM BDNF: 10 ng/ml GDNF: 10 ng/ml | After 80 days ∼100% cells positive for S100β and ∼70% GFAP-expressing cells. | GFAP, A2B5, | GLAST, GLT1, Cx43, S100β, ALDH1L1, aldolase C |
| CNTF: 10 ng/ml IGF: 10 ng/ml SHH-C: 200 ng/ml 1% FBS bFGF: 20 ng/ml | ||||||
| Sareen et al., 2014 | hiPSC | EZS | EGF: 100 ng/ml FGF2: 100 ng/ml Heparin: 5 μg/ml RA: 0.5 μM | Increased GFAP+ cells. | A2B5, Aldh1L1, GFAP | S100β, AQP4, GLAST |
| Mormone et al., 2014 | hiPSC | EB | FGF2: 10 ng/ml EGF: 20 ng/ml FGF+EGF+CNTF: 20 ng/ml Noggin: 500 ng/ml | 99% GFAP+ cell population after 28–35 days | Musashi, Nestin, A2B5 | GFAP, A2B5 |
| Caiazzo et al., 2014 | hfibroblast | Direct reprogramming | – | SOX9, Vimentin | GFAP | |
| Zhou et al., 2016 | hiPSC | EB | LDN193189: 0.2 μM SB431542: 10 μM AA: 0.2 mM | Spontaneous emergence approach: By ∼4 weeks, GFAP+ cells were quantified. | For synaptic analysis MAP2, Synapsin-1, β-III-Tubulin | GFAP, AQP4 |
There are two major approaches for differentiation of human PSCs to astrocytes: EB, monolayer culture. For both EB and monolayer approaches, stage-specific application of key growth factors (GFs) in defined media are required for optimal astrogenesis. Some protocols use fetal bovine serum (FBS) or small molecules to induce differentiation (see text for details of specific protocols). EB, embryoid body; h, human; m, mouse; AA, ascorbic acid; RA, retinoic acid; EZS, EZ spheres; ML, monolayer; NS, neurospheres. EZ spheres are similar to embryoid bodies that are passaged using tissue chopper into 200 μm spheres.