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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 ....

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 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....

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 protocols14,16. While the utility of organoid systems is growing, monolayer-based neural differentia.......

Disclosures

The authors have no conflict of interest to disclose.

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
MEF medium:
DMEM High GlucoseGibco11965-092
FBSVWR97068-08510% final concentration
Non-Essential Amino AcidsHycloneSH30238.011X final concentration
Penicillin/StreptomycinHycloneSV300101X final concentration
Ξ²-MercaptoethanolGibco21985-0231X final concentration
hiPSC medium:
Knockout DMEM/F12GibcoΒ 12660-012
Knockout Serum Replacement (KOSR)Gibco10828-02820% final concentration
Non-Essential Amino AcidsHycloneSH30238.011X final concentration
GlutamaxGibcoΒ 350500611X final concentration
Penicillin/StreptomycinHycloneSV300101X final concentration
Ξ²-Mercaptoethanol (1000X)Gibco21985-0231X final concentration
DDM medium:
Knockout DMEM/F12GibcoΒ 12660-012
Non-Essential Amino AcidsHycloneSH30238.011X final concentration
GlutamaxGibcoΒ 350500611X final concentration
Penicillin/StreptomycinHycloneSV300101X final concentration
Albumax (10%)InvitrogenΒ 11020-0210.5 X of 10% Albumax is final concentration
NPC medium:
DDM medium
N2 SupplementGibcoΒ 175020481X final concentration
B-27 Supplement (50X), minus vitamin AGibcoΒ 125870101X final concentration
Neural Differentiation medium (ND):
DDM medium1/2 of volume
Neurobasal MediumGibcoΒ 21103-0491/2 of volume
N2 SupplementGibcoΒ 175020480.5 X final concentration
B-27 Supplement (50X)GibcoΒ 175040440.5 X final concentration
GlutamaxGibcoΒ 350500611X of Neurobasal medium
Penicillin/StreptomycinHycloneSV300101X of Neurobasal medium
Antibodies and reagents for immunostaining:
ParaformaldehydeSigma-Aldrich158127-500G4%
DPBS, no calcium, no magnesiumSigma-AldrichD5652
Triton-X-100 SolutionSigma-AldrichX100-500ML0.20%
BSAHycloneA7979-50ML1.00%
Purified anti-tubulin Ξ²-3 (TUBB3) (TUJ1)BioLegend8012021:500 dilution
Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa fluor 594 conjugateLife Tech InvitrogenΒ A212031:250 dilution
Purified Mouse-Anti-Human Ki67BD Pharmingen5506091:100 dilution
Purified anti-PAX6BioLegends9013021:100 dilution
Alexa fluor 488 Donkey (anti-rabbit)Life Tech InvitrogenΒ A212061:250 dilution
DAPI Solution (5 mg/mL)SigmaD95421:1000 dilution
Others
Cell detachment solution (Accutase)Β GibcoΒ A11105-01Ready to use working solution
Rock inhibitor (RI)Β SellechckemΒ Y2763210 mM/ml final concentration
DorsomorphinΒ SellechckemΒ S73060.125 nM/ml final concentration
DPBS with calcium and magnisium (DPBS+ Ca, Mg)GibcoΒ 14040133Ready to use working solution
DPBS without calcium and magnisiumGibcoΒ 14190136Ready to use working solution
Gelatin Type ASigmaΒ G2500-100G0.10%
hESC-qualified basement membrane matrix (Matrigel GFR)Β CorningΒ 3562301 mg stock vial diluted 1:240
Trypsin 0.05%Gibco25300054
Trypan BlueGibco15250-0610.40%
Basic Fibrablast Growth Factor (bFGF)PeprotechΒ 100-18B25 ng/ml final concentration
CollagenaseΒ GibcoΒ 17104-0191mg/ml final concentration

References

  1. Chapman, R. S., Hesketh, L. J. Behavioral phenotype of individuals with Down syndrome. Ment Retard Dev Disabil Res Rev. 6 (2), 84-95 (2000).
  2. Haydar, T. F., Reeves, R. H. Trisomy 21 and early brain development. Trends Neurosci. 35 (2), 81-91 (2012).
  3. Wisniewski, K. E. Down syndrome children often have brain with maturation delay, retardation of growth, and cortical dysgenesis. Am J Med Genet Suppl. 7, 274-281 (1990).
  4. Guidi, S.Β et al. Neurogenesis impairment and increased cell death reduce total neuron number in the hippocampal region of fetuses with Down syndrome. Brai....

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