In Vitro Modeling of Down Syndrome Neurogenesis Using Human-Induced Pluripotent Stem Cells

Summary

This protocol outlines a methodology for recapitulating Down syndrome (DS) impaired neurogenesis using DS human iPSCs. The protocol identified biphasic cell cycle defect as the cause of impaired neurogenesis in Down syndrome. It provides a robust platform for understanding cellular and molecular mechanisms underlying the abnormal neurogenesis associated with DS.

Abstract

Down syndrome (DS), caused by an extra copy of chromosome 21, is a leading cause of intellectual disability. One of the key factors contributing to this intellectual disability is impaired neurogenesis observed from fetal stages onwards. To study these neurodevelopmental abnormalities, human-induced pluripotent stem cells (hiPSCs) generated using cells obtained from DS patients provide a valuable and relevant model. Here, a comprehensive protocol is described for recapitulating DS-impaired neurogenesis observed during DS fetal stages. This protocol utilizes a pair of DS-hiPSCs having three copies of chromosome 21 and its isogenic euploid hiPSCs having two copies of chromosome 21. Importantly, the protocol described here recapitulates DS-impaired neurogenesis and found that biphasic cell cycle defect, i.e., reduced proliferation of DS neural progenitor cells (NPC) during the early phase of the neurogenic stage followed by increased proliferation of DS NPC during the late phase of the neurogenic stage is the cause of DS impaired neurogenesis. Increased proliferation of DS NPC during the late phase of the neurogenic stage leads to delayed exit from the cell cycle, causing reduced generation of post-mitotic neurons from DS NPCs. This protocol includes detailed steps for the maintenance of hiPSCs, their differentiation into neural lineages displaying biphasic cell cycle defect during the neurogenic stage, and the subsequent validation of reduced neural differentiation in DS cells. By following this methodology, researchers can create a robust experimental system that mimics the neurodevelopmental conditions of DS, enabling them to explore the specific alterations in brain development caused by trisomy 21.

Introduction

Down syndrome (DS), or trisomy 21, is the most common chromosomal abnormality and the leading cause of intellectual disability (ID)¹. Impaired neurogenesis during DS fetal development is one of the causes of intellectual disability in DS². Human DS fetal studies show a reduction in brain weight and volume, reduced neurons, increased astrocytes^3,4, and abnormal distribution of neurons in layers II and IV^5,6. Additionally, the second phase of cortical development, i.e., the emergence of lamination, is both ....

Protocol

The following protocol was followed with two pairs of Down syndrome and its isogenic euploid human iPSCs. One pair was generated using the retroviral mediated delivery method of reprogramming^19, and a second pair (NSi003-A and NSi003-B) was generated using the non-integrating Sendai virus delivery method²⁰. The protocol broadly consists of two stages: The neurogenic stage (Stage 1) and the neural differentiation stage (Stage 2). Further, two phases based on the differences in the proliferation of Down syndrome and isogenic euploid cell lines, i.e., the Early and Late phases, are observed in the neurogenic stag....

Results

Singularized human iPSCs were seeded onto qualified matrix-coated dishes as single-cell suspensions, and differentiation was initiated by removing bFGF. To inhibit non-ectodermal differentiation, Dorsomorphin, a BMP signaling inhibitor, was added from DIV 2-18²¹. For further differentiation of progenitor cells from the neurogenic stage, single-cell suspensions were replated at low density on the qualified matrix (Figure 1) for an additional 6-10 weeks to observe early.......

Discussion

In this work, an efficient undirected monolayer cortical neurodifferentiation protocol for an isogenic pair of Euploid hiPSCs and DS hiPSCs is described. Since the cells are grown as monolayers, they are more exposed to culture conditions, which is not possible to the same extent as using embryoid bodies for differentiating iPSCs, which are generally used in other protocols^14,16. While the utility of organoid systems is growing, monolayer-based neural differentia.......

Materials

Name	Company	Catalog Number	Comments
MEF medium:
DMEM High Glucose	Gibco	11965-092
FBS	VWR	97068-085	10% final concentration
Non-Essential Amino Acids	Hyclone	SH30238.01	1X final concentration
Penicillin/Streptomycin	Hyclone	SV30010	1X final concentration
β-Mercaptoethanol	Gibco	21985-023	1X final concentration
hiPSC medium:
Knockout DMEM/F12	Gibco	12660-012
Knockout Serum Replacement (KOSR)	Gibco	10828-028	20% final concentration
Non-Essential Amino Acids	Hyclone	SH30238.01	1X final concentration
Glutamax	Gibco	35050061	1X final concentration
Penicillin/Streptomycin	Hyclone	SV30010	1X final concentration
β-Mercaptoethanol (1000X)	Gibco	21985-023	1X final concentration
DDM medium:
Knockout DMEM/F12	Gibco	12660-012
Non-Essential Amino Acids	Hyclone	SH30238.01	1X final concentration
Glutamax	Gibco	35050061	1X final concentration
Penicillin/Streptomycin	Hyclone	SV30010	1X final concentration
Albumax (10%)	Invitrogen	11020-021	0.5 X of 10% Albumax is final concentration
NPC medium:
DDM medium
N2 Supplement	Gibco	17502048	1X final concentration
B-27 Supplement (50X), minus vitamin A	Gibco	12587010	1X final concentration
Neural Differentiation medium (ND):
DDM medium			1/2 of volume
Neurobasal Medium	Gibco	21103-049	1/2 of volume
N2 Supplement	Gibco	17502048	0.5 X final concentration
B-27 Supplement (50X)	Gibco	17504044	0.5 X final concentration
Glutamax	Gibco	35050061	1X of Neurobasal medium
Penicillin/Streptomycin	Hyclone	SV30010	1X of Neurobasal medium
Antibodies and reagents for immunostaining:
Paraformaldehyde	Sigma-Aldrich	158127-500G	4%
DPBS, no calcium, no magnesium	Sigma-Aldrich	D5652
Triton-X-100 Solution	Sigma-Aldrich	X100-500ML	0.20%
BSA	Hyclone	A7979-50ML	1.00%
Purified anti-tubulin β-3 (TUBB3) (TUJ1)	BioLegend	801202	1:500 dilution
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa fluor 594 conjugate	Life Tech Invitrogen	A21203	1:250 dilution
Purified Mouse-Anti-Human Ki67	BD Pharmingen	550609	1:100 dilution
Purified anti-PAX6	BioLegends	901302	1:100 dilution
Alexa fluor 488 Donkey (anti-rabbit)	Life Tech Invitrogen	A21206	1:250 dilution
DAPI Solution (5 mg/mL)	Sigma	D9542	1:1000 dilution
Others
Cell detachment solution (Accutase)	Gibco	A11105-01	Ready to use working solution
Rock inhibitor (RI)	Sellechckem	Y27632	10 mM/ml final concentration
Dorsomorphin	Sellechckem	S7306	0.125 nM/ml final concentration
DPBS with calcium and magnisium (DPBS+ Ca, Mg)	Gibco	14040133	Ready to use working solution
DPBS without calcium and magnisium	Gibco	14190136	Ready to use working solution
Gelatin Type A	Sigma	G2500-100G	0.10%
hESC-qualified basement membrane matrix (Matrigel GFR)	Corning	356230	1 mg stock vial diluted 1:240
Trypsin 0.05%	Gibco	25300054
Trypan Blue	Gibco	15250-061	0.40%
Basic Fibrablast Growth Factor (bFGF)	Peprotech	100-18B	25 ng/ml final concentration
Collagenase	Gibco	17104-019	1mg/ml final concentration