Culture and Differentiation of Human PSCs
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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…
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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.
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Generation of human neurons derived from hPSC
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hiPSCs were generated from fibroblasts from SPG11 patients (SPG11-1 and SPG11-2) and control subjects (CTRL-1 and CTRL-2) as previously described (TM Express).
- Terminal medium'>differentiation of NPCs towards neuronal cells was initiated in medium'>neural medium'>differentiation medium [NDM: NIM supplemented with 20 ng/ml brain-derived neurotrophic factor , 20 ng/ml glial cell line-derived neurotrophic factor , 1 mm dibutyryl-cyclic AMP and 200 nm ascorbic acid ] at a density of 40 000 cells/cm2 on PORN/laminin-coated plates or glass coverslips.
- Neuronal cultures were kept for differentiation under these conditions from 12 to 40 days with a half medium change every week.
- Since we have used one NPC/neuronal line from each control and SPG11 patient groups, for the sake of simplicity for the readers, we will hereafter refer to the neuronal lines as CTRL-1, CTRL-2, SPG11-1 and SPG11-2 from the control and SPG11 patients, respectively.
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Dopaminergic neuronal induction of bone marrow-derived mesenchymal stem cells
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Passage 3 bone marrow-derived mesenchymal stem cells were trypsinized and subcultured onto polylysine-coated coverslips in multiwell plates.
- Each plate contained 1 × 105 cells.
- At approximately 80% confluence, bone marrow-derived mesenchymal stem cells were induced into dopaminergic neurons using different inducers as follows: in the Xiangdan injection group, cells were pre-induced in L-Dulbecco's-modified Eagle's medium containing 10% fetal bovine serum and 25 ng/mL basic fibroblast growth factor for 24 hours, followed by L-Dulbecco's-modified Eagle's medium and 20% Xiangdan injection (containing 1 000 g/L salvia miltiorrhiza, 1 000 g/L dalbergia odorifera (No. Z13021387), Hebei Tiancheng Pharmaceutical Co., Ltd., China) for 3 hours; in the all-trans retinoic acid + glial cell line-derived neurotrophic factor group, cells were induced in medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>neurobasal cells.
- At approximately 80% confluence, bone marrow-derived mesenchymal stem cells were induced into dopaminergic neurons using different inducers as follows: in the Xiangdan injection group, cells were pre-induced in L-Dulbecco's-modified Eagle's medium containing 10% fetal bovine serum and 25 ng/mL basic fibroblast growth factor for 24 hours, followed by L-Dulbecco's-modified Eagle's medium and 20% Xiangdan injection (containing 1 000 g/L salvia miltiorrhiza, 1 000 g/L dalbergia odorifera (No. Z13021387), Hebei Tiancheng Pharmaceutical Co., Ltd., China) for 3 hours; in the all-trans retinoic acid + glial cell line-derived neurotrophic factor group, cells were induced in medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>neurobasal medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium containing 25 ng/mL basic fibroblast growth factor and 2% B27 for 24 hours, followed by medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>neurobasal medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium containing 2% B27, 1 μM all-trans retinoic acid and 50 ng/mL glial cell line-derived neurotrophic factor for 6 days; in the sonic hedgehog + fibroblast growth factor 8 group, cells were induced in medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>neurobasal medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium containing 25 ng/mL basic fibroblast growth factor and 2% B27 for 24 hours, followed by medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium'>neurobasal medium'>medium'>medium'>medium'>medium'>medium'>medium'>medium containing 2% B27, 250 ng/mL sonic hedgehog and 100 ng/mL fibroblast growth factor 8 for 12 days; in the control group, cells were cultured without any inducers.
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Neural differentiation
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iPSC-derived neural progenitors were generated by either embryoid body (EB)-based or monolayer differentiation according to established protocols [
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