Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells

Abstract

Source: Cvetkovic, C., et al. Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells. J. Vis. Exp. (2018).

The video demonstrates a method to generate astrospheres from progenitors from human pluripotent stem cells. It involves the growth and proliferation of stem cells with nutrient media containing growth factors and kinase inhibitors. These cells then aggregate and differentiate into astrocytes, forming astrospheres.

Protocol

1. Cell Culture and Reagent Preparation

1. Prepare coated plates for cell culture.
   1. Dilute extracellular matrix (ECM) coating solution with DMEM/F12 media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of 3 mL each and immediately store in -20 °C. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringing the total volume to 36 mL for a concentration of 80 µg/mL.
   2. Coat 1 mL per well of a 6-well cell culture plate with ECM working solution. Add an additional 1 mL DMEM/F12 per well (if necessary) to ensure that the surfa....

Results

Figure 1: Stepwise depiction of the differentiation and formation of 3D neural spheres derived from hPSCs. (A) Timeline of key steps in the protocol. (B) Pure populations of neural cells can be generated from human induced pluripotent stem cells (hPSCs). For.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 well plate	Fisher Scientific	08-772-1B
15 ml conical tubes	Olympus Plastics	28-101
Accutase	Sigma	A6964-100ML	Detachment solution
AggreWell plate	Stemcell Technologies	34850
Anti-Adherence Rinsing Solution	Stemcell Technologies	7010	Prevent cell adhesion to microwell plates
Anti/anti	Thermofisher	15240062
B27	Thermofisher	17504044	Media Supplement
BrainPhys neuronal medium	Stemcell Technologies	5790	Neurophysiological basal medium alternative
Cryostor CS10	Stemcell Technologies	7930	Cryopreservation medium with 10% DMSO
DMEM/F12	Thermofisher	10565-042	With GlutaMAX supplement
DMH-1	Stemcell Technologies	73634	HAZARD: Toxic if swallowed. Working concentration: 2 uM
Doxycycline Hydrochloride (Dox)	Sigma	D3072-1ml	HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL
Epidermal growth factor (EGF)	Peprotech	AF-100-15	Working concentration: 10 ng/mL
Fibroblast growth factor-2 (FGF)	Peprotech	100-18B	Working concentration: 10 ng/mL
Hemacytometer or automatic cell counter	Life Technologies	AMQAX1000
Matrigel membrane matrix	Corning	354230	ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.
Mounting solution
N2	Thermofisher	17502048	Media Supplement
Phosphate buffered saline (PBS)	Stemcell Technologies	CA008-300
ReLeSR	Stemcell Technologies	5872	Detachment and passaging reagent
Rho-Kinase Inhibitor Y27632- (Y)	Tocris	1254	Working concentration: 10 uM
SB431542	Stemcell Technologies	72234	Working concentration: 2 uM
T25 Culture Flask	Olympus Plastics	25-207	Vented caps
T75 Culture Flask	Olympus Plastics	25-209	Vented caps