TITLE: NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells
AUTHORS: Jason Tchieu, Elizabeth L. Calder, Sudha R. Guttikonda, Eveline M. Gutzwiller, Kelly A. Aromolaran, Julius A. Steinbeck, Peter A. Goldstein, Lorenz Studer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The mechanistic basis of gliogenesis, which occurs late in human development, is poorly understood. Here we identify nuclear factor IA (NFIA) as a molecular switch inducing human glial competency. Transient expression of NFIA is  sufficient to trigger glial competency of human pluripotent stem cell-derived neural stem cells within 5 days and to convert these cells into astrocytes in the presence of glial-promoting factors, as compared to 3–6 months using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli and engraft in the adult mouse brain. Differentiation involves rapid but reversible chromatin remodeling, glial fibrillary acidic protein (GFAP) promoter demethylation and a striking lengthening</div><div class = "text-block">of the G1 cell cycle phase. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function. We</div><div class = "text-block">used the approach to generate astrocytes with region-specific or reactive features. Our study defines key mechanisms of the gliogenic switch and enables the rapid production of human astrocytes for disease modeling and regenerative medicine.</div></div>

[STEPS]
?. [Cell culture]
Maintain human pluripotent stem cells (both embryonic and induced) on vitronectin-coated dishes in Essential 8 (E8) medium (Thermo) as previously described.
?. [Cell culture]
Purchase induced PSCs.
?. [Cell culture]
Cells were used for differentiation between passages 30–50 and passaged twice every week. Cells were subjected to mycoplasma testing every - .
?. [Cell culture]
Maintain Neural stem cells, LTNSCs and glial progenitors on poly-l-ornithine/laminin/fibronectin-coated dishes in NSC medium consisting of N2 media with  Fibroblast growth factor 2 (FGF2),  epidermal growth factor (EGF) and 1:1,000 B27 supplement. LTNSCs were used between passages 15–20 and passaged every week.
?. [Cell culture]
Maintain Human pluripotent stem cell-derived astrocytes on poly-l-ornithine/laminin/fibronectin-coated dishes in astrocyte media consisting of N2 medium with  (R&D Systems, No. 259-HE). After sorting, CD44-positive cells were passaged every week for 4 weeks and then every other week or until astrocyte processes started to detach.
[of HB-EGF]
?. [Cell culture]
Initially maintain commercial fetal astrocytes (Sciencell) in a commercial medium containing serum (Sciencell). Switch the serum-containing medium to N2 with  for at least two passages before performing the experiments.
[HB-EGF]
?. [Cell culture]
Maintain human pluripotent stem cell-derived cortical excitatory neurons on poly-l ornithine / laminin / fibronectin-coated dishes in neurobasal medium with brain-derived neurotrophic factor (BDNF), ascorbic acid, glial cell line-derived neurotrophic factor (GDNF) and cyclic adenosine monophosphate. Change more than  of the medium every week.
?. [Differentation of hPSCs towards dorsal forebrain NSCs]
Dorsal forebrain patterning. Dissociate human PSCs into single cells, and 2.5–3.0 × 105 cells per cm2 were plated onto Matrigel (BD Biosciences)-coated dishes in E8 containing   (No. Y-27632).
[ROCK inhibitor]
?. [Differentation of hPSCs towards dorsal forebrain NSCs]
The following day (day 0), switch the cells to Essential 6 (E6) medium containing  LDN193189 (LDN, Stemgent) and  SB431542 (SB, Tocris; LSB).
?. [Differentation of hPSCs towards dorsal forebrain NSCs]
Change medium every day for nine additional days (d8) as previously described.
?. [Differentation of hPSCs towards dorsal forebrain NSCs]
To better promote an anterior forebrain fate (that is, for iPSCs), we add  of XAV939 (Stemgent) in addition to LSB for  (d0–2) then from d3–8 maintained the cells in LSB without XAV939.
?. [Generation of cortical rosettes/NSCs]
From d8, dissociate the cells with Accutase for  at  and passed through a 40 μm cell strainer.
37 °C
?. [Generation of cortical rosettes/NSCs]
Resuspend the cells in N2 medium with brain-derived neurotrophic factor (BDNF), ascorbic acid, Sonic Hedgehog (SHH) and fibroblast growth factor 8 (FGF8) (N2-BASF), and plated at 5 × 105 cells on air-dried poly-l-ornithine/laminin/fibronect in-coated plates in  droplets.
20 µl
?. [Generation of cortical rosettes/NSCs]
Incubate the droplets at  for  -  . N2-BASF8 medium +  ROCKi medium was overlaid on the droplets and the medium changed every day. Rosette formation was expected within -  (d12).
37 °C
?. [Generation of cortical rosettes/NSCs]
Following rosette formation, dissociate cells with Accutase and replated at relatively high density (3.5 × 105 cells per cm2) to prevent spontaneous differentiation. Assay the cultured cells for correct regional patterning,differentiated or frozen.
?. [Neuro-ectodermal differentiation of hPSCs towards ventral spinal cord]
Similar to the dorsal forebrain, plate 2.5–3.0 × 105 cells per cm2 onto Matrigel (BD Biosciences)-coated dishes in hPSC knockout serum replacement (KSR)-based medium containing  ROCK inhibitor and  FGF2.
?. [Neuro-ectodermal differentiation of hPSCs towards ventral spinal cord]
The following day (day 0), switch the cells from a progressive gradient of KSR to N2 medium containing  LDN193189 and  SB431542 (LSB).
?. [Neuro-ectodermal differentiation of hPSCs towards ventral spinal cord]
The following day (day 1), switch the medium to LSB with  of retinoic acid and  of purmorphamine and maintained for an additional  (d12).
?. [Neuro-ectodermal differentiation of hPSCs towards ventral spinal cord]
Dissociate spinal cord progenitors with Accutase for  at  and pass it through a 40 μm cell strainer. Plate the cells at 2.5 × 105 cells per cm2 on poly-l-ornithine/laminin/fibronectin-coated plates and assayed for correct regional patterning, differentiated or frozen.
37 °C
?. [Derivation of LTNSCs from hPSCs]
Initially, differentiated hPSCs using the method employed for dorsal forebrain NSCs. After initial regional patterning, differentiated cells were dissociated using  dispase for  .
?. [Derivation of LTNSCs from hPSCs]
Separated the cells into clumps and resuspend in N2 medium containing   FGF2 and cultured in sterile, non-tissue culture-treated dishes.
?. [Derivation of LTNSCs from hPSCs]
Cells were expected to form a high number of neurospheres, and by 3-5 days neural rosette formation within the spheres should be apparent. Having purified the neurosphere cultures, they were landed on poly-l-ornithine / laminin / fibronectin-coated plates and cultured in N2 with  FGF2,  EGF and 1:1,000 B27 supplement (NSC media).
?. [Derivation of LTNSCs from hPSCs]
Observe Rosette-stage NSC outgrowth to confluency and then pass at high density (approximately 1:3) over 2-3 months.
?. [Derivation of LTNSCs from hPSCs]
Cells maintaining neuroepithelial morphology by passage 10 in NSC media were kept and analyzed for early NSC markers and differentiation potential.
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Clone NFIA and SOX9 from cDNA using hPSC-derived astroglial progenitors (d90). Digest FUW-tetO-GFP (Addgene, No. 30130) with the restriction enzyme EcoRI to remove the GFP fragment, and either insert NFIA or SOX9 using traditional ligation cloning.
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Plasmids containing NFIA, SOX9, FUCCI-O or M2-rtTA (Addgene, No. 20342), the packaging vector psPAX2 (Addgene, No. 12260) and transfect the envelope pMD2.G (Addgene, No. 12259) into 293 T cells X-tremeGene HP (Sigma) at a molar ratio of 1:2:1.
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Harvest virus at 48 and  post-transfection and concentrate it using AMICON Ultra-15 Centrifugal Filter Units (Millipore).
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Plate NSCs at 3.5 × 105 cells per cm2 on poly-l-ornithine/laminin/fibronectin-coated dishes.
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Incubate the cells with viral particles generated (as described above) for - .
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Switch the medium to NSC medium with  -  doxycycline and daily medium change for a minimum of  .
?. [Generation of NFIA- and SOX9-inducible constructs and lentiviral production and infection]
Detach cells using  trypsin and wash several times in preparation for CD44 labeling.
?. [Intracellular FACS analysis and sorting]
For both live and fixed sorting, dissociate cells using  trypsin and washed twice with phosphate buffered saline (PBS).
?. [Intracellular FACS analysis and sorting]
Fixed GFAP and CD44 analysis.Using the BD Cytofix/Cyto Perm kit (BD), resuspend 1 × 106 cells in  Cytofix and placed   for  .
1 mL
on ice
?. [Intracellular FACS analysis and sorting]
Wash the cells twice with 1× Cyto Perm Buffer and resuspended in  of 1× Cyto Perm Buffer.
100 µl
?. [Intracellular FACS analysis and sorting]
Add Alexa 647-conjugated CD44 (Biolegend) and unconjugated GFAP (Dako) to the cells, as described by the manufacturer, and incubate for  .
on ice
?. [Intracellular FACS analysis and sorting]
Wash the cells twice with 1× Cyto Perm Buffer and resuspended in  of 1× Cyto Perm Buffer for secondary labeling.
100 µl
?. [Intracellular FACS analysis and sorting]
For labeling of GFAP, add Alexa 488 or 555 to the cells for  . Wash cells twice with 1× Cyto Perm Buffer and submit for analysis.
on ice
?. [Intracellular FACS analysis and sorting]
Live CD44 sorting:Resuspend approximately 4–5 × 106 cells in  of sort buffer ( fetal bovine serum,  EDTA in PBS), and incubate CD44 conjugated with Alexa 647, as per the manufacturer’s instructions,   for  -  .
100 µl
on ice
?. [Intracellular FACS analysis and sorting]
Wash cells twice with sort buffer and submitted to the sorter.
?. [Intracellular FACS analysis and sorting]
Maintain CD44-positive cells in astrocyte induction medium (N2 with  heparin-bound EGF (R&D Systems) and  leukemia inhibitory factor (Peprotech) without doxycycline.
?. [Immunohistochemistry]
Fix cells in PBS containing  paraformaldehyde for  , permeabilized using PBS with  Triton-X for  and stored in PBS with  Tween-20.
?. [Immunohistochemistry]
Contain the blocking solution   donkey serum in PBS with  Tween-20. Dilute primary antibodies in the blocking solution and typically incubate  at  .
4 °C
?. [Immunohistochemistry]
Conjucate Secondary antibodies to either Alexa 488, Alexa 555 or Alexa 647 (Thermo) were added to the cellswith incubation for  .
?. [Immunohistochemistry]
Identify nuclei by staining the cells with 4′,6-diamidino-2-phenylindole (DAPI, Thermo). A list of antibodies used in this study is given in Supplementary Table 3.
?. [Immunoblot]
Harvest and lyse cells with RIPA buffer, and quantify  protein using Precision Red (Cytoskeleton, Inc).
?. [Immunoblot]
Load  of protein to analyze protein expression. Full scans of blots for Fig. 2b (SupplementaryFig. 22), Fig. 4f (Supplementary Fig. 23), Fig. 4k (Supplementary Fig. 24) and Supplementary Fig. 7c (Supplementary Fig. 25) are available.
10 µg
?. [Gene expression and ATAC-sequencing analysis]
RNA sequencing. Isolate RNA as previously described. Submit total RNA to the Memorial Sloan Kettering Cancer Center (MSKCC) Genomics Core for paired-end sequencing, aiming for 30–40 million reads.
?. [Gene expression and ATAC-sequencing analysis]
Trim raw FASTQ files for adapters and aligned to the ENSEMBL GRCh38 genome build using STAR 2.5.0.
?. [Gene expression and ATAC-sequencing analysis]
Generate and import matrices from the aligned files using HTSeq into DESeq2 for further analysis using a standard pipeline.
?. [Gene expression and ATAC-sequencing analysis]
A list of the normalized read counts represented in Fig. 1 and Supplementary Fig. 8 can be found in SupplementaryTable 1, and a list of genes expressed from all samples in Fig. 4 can be found in Supplementary Table 2.
?. [Gene expression and ATAC-sequencing analysis]
ATAC sequencing:Raw FASTQ files were aligned to the hg19 genome build using Bowtie2. Perform comparative analysis of alignment using the deepTools software package. Perform motif analysis and peak annotation using HOMER software and visualized using the IGV browser.
?. [Gene expression and ATAC-sequencing analysis]
Upload all FASTQ files and Supplementary files to National Center for Biotechnology Information Gene Expression Omnibus under accession code GSE104232.
?. [Cytokine treatment of human astrocytes]
Plate astrocytes at 2 × 104 cells per cm2 and treated with  IL-1α (Sigma),  tumor necrosis factor (Cell Signaling Tech) and  C1q (MyBioSource) for  .
?. [Cytokine treatment of human astrocytes]
Isolate the medium and spun down to remove debris, and measure C3 levels using the Human Complement C3 ELISA Kit (Abcam) as per the manufacturer’s instructions.
?. [Cell cycle analysis]
Isolate cells with dissociated nuclei using resuspension buffer ( Tris-HCl,  NaCl,  MgCl2) and subsequently the resuspension buffer with  NP-40 for cell cycle analysis.
?. [Cell cycle analysis]
Add propidium iodine () to the cells followed by analysis with FACS.
?. [Cell cycle analysis]
Analyse a minimum of 10,000 events per condition. Data acquired were imported and analyzed by Flowjo software.
?. [Transplantation of NSCs, glial precursors and astrocytes into adult cortex]
Perform all surgeries according to National Institutes of Health guidelines and approve it by the local Institutional Animal Care and Use Committee, the Institutional Biosafety Committee and the Embryonic Stem Cell Research Committee. A total of eight (or 20) [CF1] NOD-SCID IL2-Rgc null mice (20–35 g; Jackson Laboratory) received cell transplantation.
?. [Transplantation of NSCs, glial precursors and astrocytes into adult cortex]
Anesthetize mice with isoflurane 5% at a maintenance flow rate of 2–3%. Transplant a total of 7 × 104 NFIA-inducedastrocytes in  through a  Hamilton syringe at a rate of  by an infusion pump attached to a stereotactic micromanipulator, into the genu of the corpus callosum (coordinates: AP +0.740, ML −1.00, DV −2.30 from bregma).
2 µl
5 µl
?. [Transplantation of NSCs, glial precursors and astrocytes into adult cortex]
Transplant a total of 2 × 105 LTNSC  into the subcortical gray matter, striatum (coordinates AP +0.500, ML −1.90, DV –3.20 from bregma).
2 µl
?. [Transplantation of NSCs, glial precursors and astrocytes into adult cortex]
Transplant a total of 7.5 × 104 H1-GFP-derived astrocytes in  into the genu of the corpus callosum (coordinates: AP +0.740, ML –1.00, DV –2.30 from bregma).
2 µl
?. [Transplantation of NSCs, glial precursors and astrocytes into adult cortex]
Sacrify the mice at 1, 6 and 12 weeks after transplantation for immunohistochemical analysis.
?. [Tissue processing]
Euthanize mice were euthanized with an overdose of pentobarbital given intraperitoneally, then transcardially perfused with PBS followed by paraformaldehyde 4%.
?. [Tissue processing]
Remove brains after gentle dissection, maintained overnight in 4% paraformaldehyde then soaked in 30% sucrose for  - .
?. [Tissue processing]
Perform brain coronal sectioning (  at ) by cryostat after embedding with Optimal Cutting Temperature (OCT) (Sakura Finetek).
30
-20 °C
?. [Calcium imaging]
Plate Human pluripotent stem cell-derived neural stem cells, astrocytes or primary astrocytes (Sciencell) onto poly-l-ornithine/laminin/fibronectin-coated  black ΔT dishes (Bioptechs) and use for calcium imaging as previously described between days 60 and 120.
?. [Calcium imaging]
Incubate Cultures with  Fura-2 (Thermo) for  at  and dishes were mounted on a ΔT Heated Lid w/Perfusion system (Bioptechs).
37 °C
?. [Calcium imaging]
Perfuse cultures with normal Tyrode’s solution () containing  NaCl,  KCl,  glucose,  HEPES,  MgCl2,  CaCl2 and  (w/v) bovine serum albumin.
?. [Calcium imaging]
Supplement cultures with glutamate ( ), ATP ( ) or KCl () for  and imaged every 30 s at 340 and 380 nm at a minimum of seven positions.
?. [Calcium imaging]
Analyse time-lapse images using FIJI (ImageJ) by calculating the signal ratio between 380 and 340 nm.
?. [Glutamate excitotoxicity assay]
Derive cortical neurons by differentiating hPSCs towards a neuro-ectodermal fate (see above). Dissociate Neuro-ectodermal cells and replate to generate neural rosettes, and further differentiate into neurons by treatment with  of a γ-secretase inhibitor (DAPT).
?. [Glutamate excitotoxicity assay]
Replate neurons and assay for maturation markers or glutamate excitoxicity with or without astrocytes.
?. [Glutamate excitotoxicity assay]
For glutamate excitotoxicity studies, plate 100,000 neurons per cm2 on poly-l-ornithine/laminin/fibronectin dishes in N2 medium with BDNF, ascorbic acid and GDNF.
?. [Glutamate excitotoxicity assay]
Add NFIA-induced astrocytes at 150,000 cells per cm2 and co-culture for an additional  .
?. [Glutamate excitotoxicity assay]
Treat cells with  or  (final) l-glutamate for  in Hanks’ buffered salt solution and recover in N2 medium with BDNF, ascorbic acid and GDNF.
100
500
?. [Glutamate excitotoxicity assay]
Add resazurin  after glutamate treatment to determine cell viability.
?. [Bisulfite conversion and sequencing]
Treat LTNSCs infected with NFIA with dox for  and sorte for CD44.
?. [Bisulfite conversion and sequencing]
Isolate cells were and perform bisulfite conversion using the EZ DNA Methylation-Direct Kit (Zymo) asdescribed by the manufacturer.
?. [Bisulfite conversion and sequencing]
Primers for the regions of the GFAP STAT3 binding site were described previously.
?. [Bisulfite conversion and sequencing]
Briefly, P1 and P2 correspond to –1,500 bp from the start site of GFAP (P1 forward: 5′ AGGAGGGTTGTTTGTTTTTTAGAA, P1 reverse: 5′ CCCTTCCTTATCTAACCTCCCTATA and P2 forward: 5′ GTAGATTTGGTAGTATTGGGTTGGT, P2 reverse: 5′ CCCTCACCCATTTATATCCTTAAA.
?. [Bisulfite conversion and sequencing]
Amplify the GFAP promoter region using ZymoTaq Premix (Zymo) and cloned into the TOPO Zero Blunt vector (Invitrogen). Sent a minimum of ten colonies per condition for sequencing.
?. [Electrophysiology]
Perform whole-cell recordings as described previously, with slight modifications. Briefly, visualize neurons using a Zeiss microscope (Axioscope) equipped with a ×4 objective and a ×40 water immersion.
?. [Electrophysiology]
Record neurons at - .
23 °C
24 °C
?. [Electrophysiology]
Measure input resistance from voltage response elicited by intracellular injection of a current pulse(−100 pA, 200 ms).
?. [Electrophysiology]
Filter low-pass membrane voltage at 5 kHz and digitize at 10 kHz, using a Multiclamp 700B amplifier connected to a DigiData 1322 A interface (Axon Instruments) using Clampex 10.2 software (Molecular Devices).Calculate liquid junction potentials and correct offline.
?. [Electrophysiology]
During recording, perfuse neurons with freshly prepared artificial cerebrospinal fluid ( NaCl,  NaHCO3,  KCl,  NaH2PO4,  MgCl2,  CaCl2 and  glucose), and saturate the solution with  O2– CO2.
?. [Electrophysiology]
Pipette solution for all recordings contained  CsCl,  NaCl,  HEPES,  EGTA,  Mg-ATP,  Na-GTP and  Na2-phosphocreatine, pH adjust to  with CsOH.
?. [Electrophysiology]
Add Bicuculline methochloride ( , Tocris),  strychnine HCl (Sigma) and  tetrodotoxin (TTX, Alomone Labs) to the artificial cerebrospinal fluid for mEPSC recordings to block gamma-aminobutyric acid receptors, glycine receptors and Na+ channels, respectively.
?. [Electrophysiology]
Hold neurons at –80 mV and continuous recording of mEPSCs was made using Axoscope software (MolecularDevices).
?. [Electrophysiology]
Perform data processing and analysis using MiniAnalysis (Synaptosoft) and Clampfit 10 (Molecular Devices). Detect Events by setting the threshold value, followed by visual confirmation of mEPSC detection.
?. [Electrophysiology]
Perform statistical analysis using Student’s t-test or Mann–Whitney rank sum test as necessary, with a significant difference at P