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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.
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.
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 astrocytes3,4, and abnormal distribution of neurons in layers II and IV5,6. Additionally, the second phase of cortical development, i.e., the emergence of lamination, is both ....
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 reprogramming19, 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....
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.......
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 protocols14,16. While the utility of organoid systems is growing, monolayer-based neural differentia.......
The authors have no conflict of interest to disclose.
The authors thank Prof. Stuart H. Orkin for providing us with a pair of Down syndrome and isogenic euploid hiPSCs. The authors are also thankful to the National Centre for Cell Science (BRIC-NCCS), Pune, for providing the funding to carry out this work.
....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 |
Ξ²-Mercaptoethanol | Gibco | 21985-023 | 1X final concentration |
hiPSC medium: | |||
Knockout DMEM/F12 | GibcoΒ | 12660-012 | |
Knockout Serum Replacement (KOSR) | Gibco | 10828-028 | 20% final concentration |
Non-Essential Amino Acids | Hyclone | SH30238.01 | 1X final concentration |
Glutamax | GibcoΒ | 35050061 | 1X final concentration |
Penicillin/Streptomycin | Hyclone | SV30010 | 1X final concentration |
Ξ²-Mercaptoethanol (1000X) | Gibco | 21985-023 | 1X final concentration |
DDM medium: | |||
Knockout DMEM/F12 | GibcoΒ | 12660-012 | |
Non-Essential Amino Acids | Hyclone | SH30238.01 | 1X final concentration |
Glutamax | GibcoΒ | 35050061 | 1X final concentration |
Penicillin/Streptomycin | Hyclone | SV30010 | 1X final concentration |
Albumax (10%) | InvitrogenΒ | 11020-021 | 0.5 X of 10% Albumax is final concentration |
NPC medium: | |||
DDM medium | |||
N2 Supplement | GibcoΒ | 17502048 | 1X final concentration |
B-27 Supplement (50X), minus vitamin A | GibcoΒ | 12587010 | 1X final concentration |
Neural Differentiation medium (ND): | |||
DDM medium | 1/2 of volume | ||
Neurobasal Medium | GibcoΒ | 21103-049 | 1/2 of volume |
N2 Supplement | GibcoΒ | 17502048 | 0.5 X final concentration |
B-27 Supplement (50X) | GibcoΒ | 17504044 | 0.5 X final concentration |
Glutamax | GibcoΒ | 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 Ξ²-3 (TUBB3) (TUJ1) | BioLegend | 801202 | 1:500 dilution |
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa fluor 594 conjugate | Life Tech InvitrogenΒ | 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 InvitrogenΒ | A21206 | 1:250 dilution |
DAPI Solution (5 mg/mL) | Sigma | D9542 | 1:1000 dilution |
Others | |||
Cell detachment solution (Accutase)Β | GibcoΒ | A11105-01 | Ready to use working solution |
Rock inhibitor (RI)Β | SellechckemΒ | Y27632 | 10 mM/ml final concentration |
DorsomorphinΒ | SellechckemΒ | S7306 | 0.125 nM/ml final concentration |
DPBS with calcium and magnisium (DPBS+ Ca, Mg) | GibcoΒ | 14040133 | Ready to use working solution |
DPBS without calcium and magnisium | GibcoΒ | 14190136 | Ready to use working solution |
Gelatin Type A | SigmaΒ | G2500-100G | 0.10% |
hESC-qualified basement membrane matrix (Matrigel GFR)Β | CorningΒ | 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) | PeprotechΒ | 100-18B | 25 ng/ml final concentration |
CollagenaseΒ | GibcoΒ | 17104-019 | 1mg/ml final concentration |
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