TITLE: Differentiation of Astrocytes from Human iPSC-derived NPCs 
AUTHORS: Balazs Szeky, Anita Feher, Melinda Zana, Radek Kucera, Jan Lochman, Andras Dinnyes

[DESCRIPTION]
Astrocytes are multifunctional glial cells of the central nervous system (CNS).  They play essential roles in the metabolic support of neurons, synaptic transmission, maintaining blood-brain-barrier and immunological homeostasis. On the other hand, distinct subtypes of astrocytes play central roles  in drug metabolism, neuroinflammation and in the exacerbation of neurodegenerative disorders. Understanding astrocye-mediated mechanisms of different CNS pathologies requires a reliable in vitro model system recapitulating in vivo astrocyte phenotypes and functionalities. To provide such models, we developed an efficient, time- and cost-effective method for the generation of functional astrocytes with high purity from human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs).

[STEPS]
SECTION: Neural Progenitor Cell Expansion
3. Thawing NPCs




SECTION: Astroglial Progenitor Cell Induction
6. Starting astrocyte induction at Day 0. Next day after the second NPC passaging, replace completed NMM intro AIM, (2mL/well for 6-well plates). Change the medium every other day at least for 21 days. Passage the APCs upon reaching confluence.



SECTION: Astrocyte Maturation
8. Initiating Astrocyte Maturation from APCs. 


SECTION: Preparation of Coated Plates and Solutions
1. Preparation of Coated Plates: 

Neural progenitor cells can be grown on poly-L-ornithine/Laminin (POL) coated plates, whereas astroglial cells and astroglial progenitors must be seeded into Matrigel coated plates. Reagents required for preparing POL and matrigel plate coatings are described below.


SECTION: Preparation of Coated Plates and Solutions
1.1. Coating of Plates With POL


 
I. Prepare the desired plate format for POL coating


II. Prepare 5-times diluted poly-L-ornithine solution in PBS.  Pipette the proper volume into the desired number of wells, incubate at 37 °C for60 min . 
III. Prepare 1 µg /cm2 laminin solution on ice . 
IV. Aspirate the Poly-L-ornithine from the plates. Wash the poly-L-ornithine-coated wells with PBS, then pipette the laminin solutions into the wells using chilled serological tips.
V. Seal the coated plates with parafilm, and store them at 4 °C overnight. Incubate the POL-coated plates at 37 °C for at least 60 min before use.

SECTION: Preparation of Coated Plates and Solutions
2. Preparation of Cell Culture Mediums
Neural maintenance medium (NMM) must be prepared for NPCs. NMM is completed with 10 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor (bFGF). 
Astrocyte induction medium (AIM) is used for astroglial progenitor cell (APC) enrichment from NPCs. 
Astrocyte maturation medium (AMM)  is used for astrocyte maturation from APCs. AMM is completed with 20 ng/mL ciliary neurotrophic factor (CNTF).

SECTION: Preparation of Coated Plates and Solutions
2.1. Preparing NMM

Mix the components of NMM (listed in the table below) in a bottletop filter using filtered pipette tips and serological pipettes. Apply vacuum tube to the filter, turn on vacuum pump and sterile filter the solution.  store NMM at 4 °C . 

 

SECTION: Preparation of Coated Plates and Solutions
2.2. Preparing AIM

Mix the components of AIM (listed in the table below) in a bottletop filter using filtered pipette tips and serological pipettes. Apply vacuum tube to the filter, turn on vacuum pump and filter the solution. Store AIM  at 4 °C . 

 

SECTION: Preparation of Coated Plates and Solutions
2.3. Preparing AMM

Mix the components of AMM (listed in the table below) in a bottletop filter using filtered pipette tips and serological pipettes. Apply vacuum tube to the filter, turn on vacuum pump and filter the solution. Store AMM at 4 °C . 


 


SECTION: Preparation of Coated Plates and Solutions
1.2. Coating of Plates With Matrigel

hESC qualified Matrigel (Corning, #CLS354277 ) must be kept on ice to prevent condensation into gel phase. Matrigel stock solutions can be used only when the solution becomes fully liquid. Matrigel must be diluted in ice-cold DMEM/F12 (Gibco, #11330032) medium.

I. Prepare the desired plate format for Matrigel coating


 
II. Prepare Matrigel solution and DMEM/F12 on ice . Add 25 ul of matrigel into every 1.5 mL of DMEM/F12.  
III. Pipette the prepared Matrigel working solution into the wells of the plate(s) using chilled serological tips. Incubate the plates at 37 °C  for at least 30 min before use. For extended storage, seal the plates with parafilm and store at 4 °C . Plates can be stored up to 14 days.

3.1. Prepare POL-coated plates and NMM before thawing NPCs. Prepare 5 mL  NMM into a 15 mL Falcon tube.

4. Passaging NPCs
3.2. Thaw one vial of frozen NPCs under sterile cell culture hood in sterile gloves. Pipette the cell suspension gently into the falcon tube containing the 5 mL NMM.
3.3. Centrifuge the cell suspension: 1000 rpm, 3 min . Discard the supernatant. 

3.4. Resuspend the pellet in NMM, complete with EGF and bFGF. Pipette the suspension into the wells of the POL-coated plate

3.5. Put the plate into a cell culture incubator, (37 °C , 5% CO2). Change medium next day to complete NMM. 

3.6. Change medium every other day onwards. Passage NPCs upon reaching confluence.
4.1. Prepare coated POL plate, complete NMM and Accutase atRoom temperature  for passaging of NPCs. 

 
5. Plating for Astrocyte Induction
4.2. Wash the cells with PBS, (2 mL /well for 6-well plates). Avoid scratching the cell monolayer.

4.3. Add Accutase, (1 mL /well for 6-well plates). Incubate at37 °C  for 3 min . After the incubation, the rollup of the cells can be observed under the microscope.

4.4. Add NMM (2 mL /well for 6-well plates) to stop the accutase digestion. Dissociate the monolayer by  pipetting. Transfer the cell suspension into the Falcon tube. 

4.5. Centrifuge the cell suspension: 1000 rpm, 3 min . Discard the supernatant, except 200 µL . Resuspend the cells. 

4.6. Add completed NMM . Perform cell counting.  Dilute the cell suspension with complete NMM to obtain 75.000-100.000 cells/cm2  (depending on further experiments) for each well of the POL-coated plate, (1 million cells/well for 6-well plates). 

4.7. Pipette the cell suspension into the POL-coated plates, (2 mL /well for 6-well plates). Distribute the cells by gentle shaking. Put the plates into a sterile cell culture incubator, (37 °C , 5% CO2).

4.8. Change the medium next day to complete NMM. Change medium every other day onwards until the next passage.
5.1. By the second passaging of the NPCs, prepare Matrigel-coated plates, AIM and Accutase.
5.2. Passage and plate cells as described in steps 4.1-4.6.  
5.3. Perform cell counting.  Dilute the cell suspension with complete NMM to obtain 25.000-75.000 cells/cm2 for each well of the Matrigel-coated plate.

5.4. Pipette the suspension into the matrigel coated plate (2mL/well for 6-well plates). Distribute the cells by gentle shaking. Put the plate into a sterile cell culture incubator, (37 °C , 5% CO2).
SECTION: Astroglial Progenitor Cell Induction
7. Passaging APCs
SECTION: Astroglial Progenitor Cell Induction
7.1. Prepare Matrigel-coated plate, AIM and Accutase atRoom temperature  for passaging of APCs. 
SECTION: Astroglial Progenitor Cell Induction
7.2. Wash the cells with PBS, (2 mL /well for 6-well plates). Aspirate the PBS. Avoid scratching the cell monolayer.
SECTION: Astroglial Progenitor Cell Induction
7.3. Add Accutase, (1 mL /well for 6-well plates). Incubate at37 °C  for 3 min -7 min until the cells begin to roll up.
SECTION: Astroglial Progenitor Cell Induction
7.4. Add AIM (2 mL /well for 6-well plates) to stop the accutase digestion. Dissociate the monolayer  and add the 1mL cell suspension into a 15 mL Falcon tube. 

SECTION: Astroglial Progenitor Cell Induction
7.5. Wash with 1 mL  AIM and collect the rest of the cell suspension into the falcon tube. 

SECTION: Astroglial Progenitor Cell Induction
7.6. Centrifuge the cell suspension: 1000 rpm, 3 min . Discard the supernatant, except 200 µL . Resuspend the cells using 200 uL pipettes with filtered tips. 
SECTION: Astroglial Progenitor Cell Induction
7.7.  Perform cell counting.  Dilute the cell suspension with AIM to obtain 25.000-75.000 cells/cm2 for each well of the Matrigel-coated plate, (0.5 million cells/well for 6-well plates). 

SECTION: Astroglial Progenitor Cell Induction
7.8. Pipette the cell suspension into the Matrigel-coated plates. Distribute the cells by gentle shaking. Put the plates into a sterile cell culture incubator, (37 °C , 5% CO2).

SECTION: Astroglial Progenitor Cell Induction
7.9. Change the medium next day to AIM. Change medium every other day onwards until the next passage.
SECTION: Astrocyte Maturation
8.1. On the 21st day of differentiation, prepare Matrigel-coated plates, AIM, AMM and Accutase for the passaging of APCs.
SECTION: Astrocyte Maturation
8.2. Passage APCs as described in steps 7.1-7.9. Change medium to AMM completed with 20 ng/mL CNTF (2 mL/well for 6-well plates) next day, and every other day onwards at least for 42 days.