Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein

Summary

Here we describe a protocol that facilitates the differentiation of human induced pluripotent stem cells into functional forebrain-specific astrocytes. This enables investigations into the role of glial cells in the pathogenesis of neurodevelopmental disorders, such as Fragile X Syndrome, and modeling of other brain disorders.

Abstract

Fragile X syndrome (FXS), a leading inherited cause of autism spectrum disorder and intellectual disability, has been studied extensively using rodent models. More recently, human stem cell-derived model systems have also been used to gain mechanistic insights into the pathophysiology of FXS. However, these studies have focused almost exclusively on neurons. Further, despite growing evidence for a key role of glia in neuronal function in health and disease, little is known about how human astrocytes are affected by FXS.

Therefore, in this study, we successfully developed a protocol that captures key spatiotemporal milestones of brain development and aligns with the process of gliogenesis as well. Together this offers a useful framework for studying neurodevelopmental disorders. First, we patterned the human induced pluripotent stem cells into the neuroectodermal lineage with dual Suppressor of Mothers against Decapentaplegic (SMAD) inhibition and small molecules. Subsequently, we utilized specific growth factors and cytokines to generate control (CTRL) and FXS patient-derived astrocytic progenitor cells (APCs). Treatment of APCs with ciliary neurotrophic factor, a differentiating cytokine, regulated and drove the progenitor cells towards astrocytic maturation, yielding forebrain-specific glial fibrillary acidic protein-expressing astrocytes.

We found that these astrocytes are functional, as evidenced by their calcium responses to ATP application, and they exhibit dysregulated glycolytic and mitochondrial metabolism in FXS. Taken together, these findings provide a useful experimental platform of human origin for the investigation of cell-autonomous and non-cell-autonomous consequences of alterations in astrocytic function caused by neurodevelopmental disorders.

Introduction

Fragile X Syndrome (FXS), a common inherited form of intellectual disability and autism spectrum disorder (ASD), is caused by the lack of fragile X messenger ribonucleoprotein (FMRP) produced by the fragile X messenger ribonucleoprotein 1 (FMR1) gene (OMIM: #300624, https://www.omim.org/entry/300624). FMRP plays a role in the regulation of mRNA translation, mRNA granule formation and transport, and microRNA-mediated regulation of gene expression¹. Thus, loss of FMRP impacts not just brain development but also adult brain function. Both mRNA transcript levels of FMR1 and immunostaining for FMRP in the brai....

Protocol

All experiments using human induced pluripotent stem cells (hiPSCs) (Table 1) were performed after obtaining appropriate institutional regulatory approvals. Figure 1A represents complete differentiation protocol of hiPSCs to mature forebrain-specific astrocytes.

1. Maintenance and expansion of hiPSCs

1. One day before plating the iPSCs, coat a 6-well dish with 1:60 diluted Matrigel (an Extracellular Matrix [ECM]) in Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 (DMEM/F12) and store at 2-8 °C.
   NOTE: Avoid storing Matrigel-coated dishes for ....

Results

Human induced pluripotent stem cell (hiPSC) colonies were maintained using commercially available defined medium and immunostained for pluripotency markers, Oct4 and Nanog (Figure 1B). FXS and CTRL APCs showed comparable highly enriched proportions of cells immunopositive for vimentin and NFIA (Figure 1C,D). We found that APCs derived from healthy and FXS hiPSCs showed substantial upregulation of FOXG1 c.......

Discussion

Here, we describe a method to generate human iPSC-derived astrocytes that serve as an assay platform for characterizing functional changes induced by FXS. These astrocytes are functionally viable in culture and exhibit various properties, as evidenced by a range of measurements carried out in the present study. A critical step in this protocol is the initial conversion of iPSCs to corticospheres using the enzymatic lifting method. At this stage, optimizing the incubation time for collagenase type IV and dispase is crucia.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Thioglycerol	Sigma-Aldrich	M6145
Accutase solution	Sigma-Aldrich	A6964	Enzyme cell detachment medium
Adenosine 5′-triphosphate magnesium salt	Sigma-Aldrich	A9187
Advanced DMEM/F-12	ThermoFisher Scientific	12634010
Antibiotic-Antimycotic (100x)	ThermoFisher Scientific	15240062
B-27 Supplement (50x), serum-free	ThermoFisher Scientific	17504044
Bovine Serum Albumin	Sigma-Aldrich	A9418
Chemically Defined Lipid Concentrate	ThermoFisher Scientific	11905031
Collagenase, Type IV, powder	ThermoFisher Scientific	17104019
Deoxyribonuclease I	Worthington Biochemical Corporation	LK003170
Dimethyl sulfoxide	Sigma-Aldrich	D2650
Dispase II, powder	ThermoFisher Scientific	17105041
DPBS, no calcium, no magnesium	ThermoFisher Scientific	14190144
Essential 8 Medium	ThermoFisher Scientific	A1517001
FXS1, FXS2	Coriell Institute of Medical Research	GM07072, GM05848	FXS patient cells
GlutaMAX Supplement	ThermoFisher Scientific	35050061	glutamine substitute
Ham's F-12 Nutrient Mix	ThermoFisher Scientific	11765054
Healthy control cells	Cedars-Sinai Medical Center	ND30625	healthy control cells
Heparin sodium salt from porcine intestinal mucosa	Sigma-Aldrich	H3149
IMDM	ThermoFisher Scientific	12440053
Insulin, human	Roche	11376497001
LDN 193189	Stratech Scientific	S2618-SEL
Leukemia Inhibitory Factor human	Sigma-Aldrich	L5283
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix	Corning	354230
MEM Non-Essential Amino Acids Solution (100x)	ThermoFisher Scientific	11140050
Mouse FGF-basic (FGF-2/bFGF) Recombinant Protein	Peprotech	450-33
Mr. Frosty freezing container	ThermoFisher Scientific	5100-0001	cryobox
N-2 Supplement (100x)	ThermoFisher Scientific	17502048
N-Acetyl-L-cysteine	Sigma-Aldrich	A9165
Neurobasal Medium	ThermoFisher Scientific	21103049
Nunc Biobanking and Cell Culture Cryogenic Tubes	ThermoFisher Scientific	377267
Nunc Cell-Culture Treated 6 well dish	ThermoFisher Scientific	140675
Papain Dissociation System	Worthington Biochemical Corporation	LK003150
Penicillin-Streptomycin	ThermoFisher Scientific	15140122
Recombinant Human CNTF Protein, CF	R&D Systems	257-NT-010
Recombinant Human EGF Protein, CF	R&D Systems	236-EG-01M
RevitaCell Supplement (100x)	ThermoFisher Scientific	A2644501
SB431542	Tocris	1614
Seahorse XFe24 Analyzer	Agilent Technologies
Seahorse XF Cell Mito Stress Test Kit	Agilent Technologies	103015-100
Seahorse XF Glycolysis Stress Test Kit	Agilent Technologies	103020-100
Tissue Culture Dishes-100 cm	Biostar Lifetech	TCD000100
Transferrin	Roche	10652202001
VWR Razor Blades	VWR International	55411-050
Antibodies
Primary antibody	Company	Catalog number	Dilution
Oct4 (C-10)	Santa Cruz Biotechnology	sc-5279	Dilution: 1:250 Secondary antibody: Goat anti-Mouse IgG, Alexa Fluor 568
Nanog	R & D Systems	AF1997	Dilution: 1:100 Secondary antibody: Donkey anti-Goat IgG, Alexa Fluor 488
Vimentin	Abcam	Ab5733	Dilution: 1:500 Secondary antibody: Goat anti-Chicken IgY, Alexa Fluor 488
NFIA	Abcam	Ab41851	Dilution: 1:500 Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 568
GFAP-cy3	Sigma	C9205	Dilution: 1:500 Secondary antibody: NA
GFAP	DAKO	Z0334	Dilution: 1:500 Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 568
S100β	DAKO	IR504	Dilution: 1:500 Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 488
Anti-Nuclei Antibody, clone 235-1	Merck Millipore	MAB1281	Dilution: 1:1000 Secondary antibody: Goat anti-Mouse IgG1, Alexa Fluor 555
Secondary antibodies			Dilution
Goat anti-Mouse IgG, Alexa Fluor 568	Thermo Fisher Scientific	A11004	1:1000
Donkey anti-Goat IgG, Alexa Fluor 488	Thermo Fisher Scientific	A11055	1:1000
Goat anti-Chicken IgY, Alexa Fluor 488	Thermo Fisher Scientific	A11039	1:1000
Goat anti-Rabbit IgG, Alexa Fluor 568	Thermo Fisher Scientific	A11011	1:1000
Goat anti-Rabbit IgG, Alexa Fluor 488	Thermo Fisher Scientific	A11034	1:1000
Goat anti-Mouse IgG1, Alexa Fluor 555	Thermo Fisher Scientific	A21127	1:1000