Neural induction
-
After 6-8 (early) and 20-30 (late) passages, iPSC and late-passage (30-40) ESCs were subjected to neural differentiation according to a previously established procedure for ESCs (Figure 4 (iPSC) or 5 × 104 (ESC) cells per ml to allow embryoid body (EB) formation.
- Differentiation medium was changed at day 3.
- On day 5, EBs were plated en bloc on tissue culture plates or chamber slides double-coated with poly-D-lysine (200 μg/ml) and mouse laminin (10 μg/ml) at a density of 1-2 × 102 EBs per cm2 in fresh medium.
- Before plating, EB were imaged to assess size and shape.
- At least 50 EBs were analyzed using MetaMorph software to determine the average EB diameter for each biological replicate.
- Twenty-four-thirty-six hours post plating, the medium was changed to neural induction medium containing GMEM, 1% N2, 2 mM glutamine, 1 mM sodium pyruvate,…
-
After 6-8 (early) and 20-30 (late) passages, iPSC and late-passage (30-40) ESCs were subjected to neural differentiation according to a previously established procedure for ESCs (Figure 4 (iPSC) or 5 × 104 (ESC) cells per ml to allow embryoid body (EB) formation.
- Differentiation medium was changed at day 3.
- On day 5, EBs were plated en bloc on tissue culture plates or chamber slides double-coated with poly-D-lysine (200 μg/ml) and mouse laminin (10 μg/ml) at a density of 1-2 × 102 EBs per cm2 in fresh medium.
- Before plating, EB were imaged to assess size and shape.
- At least 50 EBs were analyzed using MetaMorph software to determine the average EB diameter for each biological replicate.
- Twenty-four-thirty-six hours post plating, the medium was changed to neural induction medium containing GMEM, 1% N2, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 0.01% penicillin streptomycin and 10 ng/ml brain-derived neurotrophic factor (BDNF) .
- Neural induction cultures were maintained for 3, 7 or 15 days before cells were harvested for RNA extraction, electrophysiological recordings, flow cytometry analysis, or fixation with 4% paraformaldehyde for immunocytochemistry.
|
Acute BDNF treatment induces maturation of the dendritic spine population.
-
11 DIV hippocampal neurons were transfected with a vector expressing Lifeact-ruby, and 24 hrs later, the neurons were treated with vehicle or 100 ng/ml BDNF followed by time-lapse imaging every 5 minutes for 1 hr.
|
Neural precursor culture and viral transduction
-
To differentiate NPs to neurons, NPs were plated on polyornithine and laminin coated plates in NP media and grown until they reached 70% confluence.
- FGF was removed and NP media supplemented with 20 ng/ml BDNF , 20 ng/ml GDNF and 0.5 mM dibutyryl cAMP (N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt).
- Medium was exchanged every 2–3 days.
|
Monolayer neuronal differentiation
-
iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
- The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
- kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
- After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing…
-
iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
- The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
- kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
- After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing the same ligands over 7 days (75% medium'>medium'>KSR and 25% medium'>N2B27 first day, 50% of each third day, and 25% medium'>medium'>KSR with 75% medium'>N2B27 fifth day).
- Once established in N2B27 conditions, Wnt1, Noggin, SB431542 and −1 BDNF , 0.2 mM ascorbic acid and 100 ng ml−1 FGF8 were added.
- Three days later, cells were dissociated with Hank's buffered saline solution for 1 h at room temperature, and lifted mechanically, then replated en bloc on to poly-l-ornithine/laminin-coated plasticware.
- Neuronal maturation ensued with BDNF and ascorbic acid as before, supplemented with 10 ng ml−1 GDNF , 1 ng ml−1 TGFβ3 and 0.5 mM dibutyryl-cAMP for the next 7 days.
- After a total of 23–31 days, the resulting neuronal cultures were analysed by immunocytochemistry, qPCR and western blot.
|
Preparation of Primary Hippocampal Cultures
-
Primary hippocampal cultures were prepared from wild-type neonatal (E19) rat embryos (timed pregnant Sprague Dawley rats were obtained from Charles River Laboratories, Wilmington, MA) as described previously
|
Developmental regulation of BDNF-induced TrkB receptor phosphorylation.
-
(b) Representative blots of experiments showing that BDNF (50 ng/ml, 15 min, 37°C) readily induces TrkB phosphorylation at sites Y515 and Y705/6 in P8 hippocampal microslices whereas neither site is effectively phosphorylated by BDNF in P24 hippocampal microslices.
|
Immunoisolation and Culture of RGC from Postnatal Mouse Retinae
-
Seven days old mice were killed according to institutional guidelines.
- To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
- The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
- Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
- Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
- Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
- Positive cell isolation was then achieved by incubating the filtrated…
-
Seven days old mice were killed according to institutional guidelines.
- To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
- The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
- Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
- Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
- Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
- Positive cell isolation was then achieved by incubating the filtrated supernatant for 45 min on a 100-mm diameter petri dish sequentially pre-coated with goat anti-mouse-IgG and anti-Thy1.2 (mouse IgM, clone F7D5, , ).
- Non-adherent cells were thoroughly washed off, and the bound cells were released by trypsination (12,000 U/ml in Hank’s buffered salt solution [HBSS] for 10 min in 5% CO2 at 37°C) and resuspended in culture medium.
- The average yield, in thousands, of RGC per animal was 27.8 (±3.3), n = 11.
- RGC were plated at 600 cells mm−2 on glass coverslips (10 mm in diameter) centered on 12-well tissue culture plates , which were pre-coated with 5 μg/ml poly-d-lysine (MW–40 kDa).
- Neurons were cultured for a week at 37°C, 5% CO2, 95% humidity in medium'>Neurobasal medium supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and pyruvate (1 mM), glutamine (2 mM), N-acetyl-l-cysteine (60 μg/ml), putrescine (16 μg/ml), selenite (40 ng/ml), bovine serum albumin (100 μg/ml; fraction V, crystalline grade), triiodothyronine (40 ng/ml), holotransferrin (100 μg/ml), dibutyryl cyclic AMP (250 μM), insulin (5 μg/ml), progesterone (62 ng/ml), B27 (1:50), d-mannose (50 μM), brain-derived neurotrophic factor (BDNF; 25 ng/ml, , ), ciliary neurotrophic factor (CNTF; 10 ng/ml) and forskolin (10 μM).
- Cell viability was checked by examining the cell cultures under a phase contrast microscope (AxioVert25, Carl Zeiss, Jena, Germany) and determining cell adherence to the cover slip and the fraction of cells developing neurites.
- We waited a week in order to let RGC develop a massive neuritic arborisation.
- Since we were interested in the effect of PEDF on neuronal survival rather than on neurite growth, only preparations exhibiting an important development of neurites were selected for further tests.
|
Immunoisolation and Culture of RGC from Postnatal Mouse Retinae
-
Seven days old mice were killed according to institutional guidelines.
- To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
- The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
- Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
- Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
- Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
- Positive cell isolation was then achieved by incubating the filtrated…
-
Seven days old mice were killed according to institutional guidelines.
- To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
- The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
- Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
- Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
- Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
- Positive cell isolation was then achieved by incubating the filtrated supernatant for 45 min on a 100-mm diameter petri dish sequentially pre-coated with goat anti-mouse-IgG and anti-Thy1.2 (mouse IgM, clone F7D5, , ).
- Non-adherent cells were thoroughly washed off, and the bound cells were released by trypsination (12,000 U/ml in Hank’s buffered salt solution [HBSS] for 10 min in 5% CO2 at 37°C) and resuspended in culture medium.
- The average yield, in thousands, of RGC per animal was 27.8 (±3.3), n = 11.
- RGC were plated at 600 cells mm−2 on glass coverslips (10 mm in diameter) centered on 12-well tissue culture plates , which were pre-coated with 5 μg/ml poly-d-lysine (MW–40 kDa).
- Neurons were cultured for a week at 37°C, 5% CO2, 95% humidity in medium'>Neurobasal medium supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and pyruvate (1 mM), glutamine (2 mM), N-acetyl-l-cysteine (60 μg/ml), putrescine (16 μg/ml), selenite (40 ng/ml), bovine serum albumin (100 μg/ml; fraction V, crystalline grade), triiodothyronine (40 ng/ml), holotransferrin (100 μg/ml), dibutyryl cyclic AMP (250 μM), insulin (5 μg/ml), progesterone (62 ng/ml), B27 (1:50), d-mannose (50 μM), brain-derived neurotrophic factor (BDNF; 25 ng/ml, , ), ciliary neurotrophic factor (CNTF; 10 ng/ml) and forskolin (10 μM).
- Cell viability was checked by examining the cell cultures under a phase contrast microscope (AxioVert25, Carl Zeiss, Jena, Germany) and determining cell adherence to the cover slip and the fraction of cells developing neurites.
- We waited a week in order to let RGC develop a massive neuritic arborisation.
- Since we were interested in the effect of PEDF on neuronal survival rather than on neurite growth, only preparations exhibiting an important development of neurites were selected for further tests.
|
Culture and Differentiation of Human PSCs
-
Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
- The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
- Culture medium was changed every day and cells were passaged every 2–3 days.
- If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
- For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
- Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
- Media changes were…
-
Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
- The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
- Culture medium was changed every day and cells were passaged every 2–3 days.
- If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
- For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
- Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
- Media changes were performed every two days.
- For differentiation to midbrain dopaminergic (A) neurons and spinal motor neurons, hNPCPSA-NCAM+ were treated with 200 ng/ml Shh and 100 ng/ml FGF8 or 100 ng/ml Shh and 0.5 µM retinoic acid (A) for 7 or 8 days, respectively.
- The cells were re-plated on new Matrigel-coated dishes at a low density and cultured in differentiation media supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) , and 10 ng/ml glial cell-derived neurotrophic factor (GDNF) for 3 weeks.
|
Culture and Differentiation of Human PSCs
-
Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
- The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
- Culture medium was changed every day and cells were passaged every 2–3 days.
- If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
- For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
- Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
- Media changes were…
-
Human ESCs [H9 (WA09, P35–45), were obtained from WiCell Inc., Madison, WI, ] 5 cells/cm2 in medium'>medium'>N2B27 medium'>medium or 1× N2, 0.5× B27 and 0.5× G21 supplement (referred to as medium'>NBG medium'>medium) plus 20 ng/ml of basic FGF.
- The PSA-NCAM-negative cells were also cultured in the same condition as the culture of PSA-NCAM-positive other than the density of 1×105 cells/cm2.
- Culture medium was changed every day and cells were passaged every 2–3 days.
- If cells did not display sufficient purity, an additional round of cell sorting was performed to enhance the homogeneity of hNPCPSA-NCAM+.
- For further differentiation, hNPCPSA-NCAM+ were seeded at a low density (>2×104 cells/cm2) and cultured in differentiation media for 3 weeks.
- Differentiation media was composed of N2B27 or NBG medium supplemented with 0.1% fetal bovine serum and 200 µM of ascorbic acid .
- Media changes were performed every two days.
- For differentiation to midbrain dopaminergic (A) neurons and spinal motor neurons, hNPCPSA-NCAM+ were treated with 200 ng/ml Shh and 100 ng/ml FGF8 or 100 ng/ml Shh and 0.5 µM retinoic acid (A) for 7 or 8 days, respectively.
- The cells were re-plated on new Matrigel-coated dishes at a low density and cultured in differentiation media supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) , and 10 ng/ml glial cell-derived neurotrophic factor (GDNF) for 3 weeks.
|
