Immortalized striatal precursor neurons from Huntington's disease patient-derived iPS cells as a platform for target identification and screening for experimental therapeutics.
We have previously established induced pluripotent stem cell (iPSC) models of Huntington's disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders.
Pubmed ID: 34296279 RIS Download
Research resources used in this publication
- Mouse Anti-MAP-2 (Microtubule Associated Protein, MAP-2A and -2B) Monoclonal antibody, Unconjugated, Clone ap20 (RRID:AB_94856) (antibody)
- DARPP-32 (H-62) (RRID:AB_639000) (antibody)
- pBABE-neo-hTERT (RRID:Addgene_1774) (plasmid)
- pBABE GFP (RRID:Addgene_10668) (plasmid)
- pBabepuro-myc-ER (RRID:Addgene_19128) (plasmid)
Additional research tools detected in this publication
None foundAntibodies used in this publication
- Anti-rabbit IgG (H+L), F(ab) 2 Fragment (Alexa Fluor 555 Conjugate) (RRID:AB_10694110)
- Anti-mouse IgG (H+L), F(ab) 2 Fragment (Alexa Fluor 488 Conjugate) (RRID:AB_10694704)
- Rabbit Anti-GABA Antibody, Unconjugated (RRID:AB_477652)
- Bcl-11B (D6F1) XP® Rabbit mAb (RRID:AB_2797823)
- DARPP-32 (H-62) (RRID:AB_639000)
- Neuron Specific Class III Beta-Tubulin (RRID:AB_2737114)
- Mouse Anti-MAP-2 (Microtubule Associated Protein, MAP-2A and -2B) Monoclonal antibody, Unconjugated, Clone ap20 (RRID:AB_94856)
- nestin (10c2) (RRID:AB_627994)
- Mouse Anti-Human Meis2 Monoclonal Antibody, Unconjugated, Clone 63-T (RRID:AB_2143037)
- GS Homeobox 2 (GSH2) Rabbit anti-Human Polyclonal Antibody (RRID:AB_2116503)
- Anti-β-Actin Antibody (RRID:AB_476744)
- CaMKIV Antibody (RRID:AB_2068389)
- Calbindin (D1|4Q) XP Rabbit mAB (RRID:AB_2687400)
- NeuroD (D90G12) Rabbit mAb (RRID:AB_10859914)
- Monoclonal Anti-Polyglutamines antibody produced in mouse (RRID:AB_532270)
- Mouse Anti-Huntingtin Protein, aa 181-810 Monoclonal antibody, Unconjugated, Clone 1hu-4c8 (RRID:AB_2123255)
Associated grants
- Agency: NINDS NIH HHS, United States
Id: R21 NS083365 - Agency: NINDS NIH HHS, United States
Id: R01 NS086452 - Agency: NINDS NIH HHS, United States
Id: R21 NS104320 - Agency: NIGMS NIH HHS, United States
Id: T32 GM087237 - Agency: NHGRI NIH HHS, United States
Id: T32 HG009495 - Agency: NINDS NIH HHS, United States
Id: R21 NS109412 - Agency: NIGMS NIH HHS, United States
Id: R25 GM109441 - Agency: NINDS NIH HHS, United States
Id: R01 NS089076
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This is a list of tools and resources that we have found mentioned in this publication.
Anti-rabbit IgG (H+L), F(ab) 2 Fragment (Alexa Fluor 555 Conjugate) (antibody)
RRID:AB_10694110
This unknown targets rabbit IgG (H+L) F(ab) 2 Fragment
View all literature mentionsAnti-mouse IgG (H+L), F(ab) 2 Fragment (Alexa Fluor 488 Conjugate) (antibody)
RRID:AB_10694704
This unknown targets IgG (H+L)
View all literature mentionsRabbit Anti-GABA Antibody, Unconjugated (antibody)
RRID:AB_477652
This unknown targets GABA
View all literature mentionsBcl-11B (D6F1) XP® Rabbit mAb (antibody)
RRID:AB_2797823
This monoclonal targets Bcl-11B
View all literature mentionsDARPP-32 (H-62) (antibody)
RRID:AB_639000
This polyclonal targets DARPP-32 (H-62)
View all literature mentionsNeuron Specific Class III Beta-Tubulin (antibody)
RRID:AB_2737114
This monoclonal targets Tuj 1
View all literature mentionsMouse Anti-MAP-2 (Microtubule Associated Protein, MAP-2A and -2B) Monoclonal antibody, Unconjugated, Clone ap20 (antibody)
RRID:AB_94856
This monoclonal targets MAP-2 (Microtubule Associated Protein, MAP-2A and -2B)
View all literature mentionsnestin (10c2) (antibody)
RRID:AB_627994
This monoclonal targets nestin (10c2)
View all literature mentionsMouse Anti-Human Meis2 Monoclonal Antibody, Unconjugated, Clone 63-T (antibody)
RRID:AB_2143037
This monoclonal targets MEIS2
View all literature mentionsGS Homeobox 2 (GSH2) Rabbit anti-Human Polyclonal Antibody (antibody)
RRID:AB_2116503
This polyclonal targets GSX2
View all literature mentionsAnti-β-Actin Antibody (antibody)
RRID:AB_476744
This monoclonal targets slightly modified β-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys, conjugated to KLH
View all literature mentionsCaMKIV Antibody (antibody)
RRID:AB_2068389
This polyclonal targets CAMK4
View all literature mentionsCalbindin (D1|4Q) XP Rabbit mAB (antibody)
RRID:AB_2687400
This monoclonal targets Calbindin
View all literature mentionsNeuroD (D90G12) Rabbit mAb (antibody)
RRID:AB_10859914
This monoclonal targets NeuroD (D90G12) Rabbit mAb
View all literature mentionsMonoclonal Anti-Polyglutamines antibody produced in mouse (antibody)
RRID:AB_532270
This monoclonal targets Polyglutamines
View all literature mentionsMouse Anti-Huntingtin Protein, aa 181-810 Monoclonal antibody, Unconjugated, Clone 1hu-4c8 (antibody)
RRID:AB_2123255
This monoclonal targets Huntingtin Protein, aa 181-810
View all literature mentionsMouse Anti-MAP-2 (Microtubule Associated Protein, MAP-2A and -2B) Monoclonal antibody, Unconjugated, Clone ap20 (antibody)
RRID:AB_94856
This monoclonal targets MAP-2 (Microtubule Associated Protein, MAP-2A and -2B)
View all literature mentionsDARPP-32 (H-62) (antibody)
RRID:AB_639000
This polyclonal targets DARPP-32 (H-62)
View all literature mentions
