Huntington's Disease (HD) In Vitro Modeling Service

Huntington's Disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene, leading to the production of mutant huntingtin (mHTT) protein. This results in progressive neuronal loss, particularly in the striatum and cortex, manifesting as motor dysfunction, cognitive decline, and psychiatric symptoms.

Creative Biolabs has successfully developed a series of cellular and animal HD models and is capable of performing specific assays to screen compounds, RNAi and small molecules to find a novel effective cure or treatment to slow down or stop the disease's progression.

Available In Vitro Models

• Human iPSC-derived Models

Human induced pluripotent stem cell (iPSC)-derived neural cells are rapidly transforming HD research and drug discovery. We offer a comprehensive suite of iPSC-derived models, including CRISPR-Cas9 gene-edited glutamatergic neurons with 50 CAG repeats in the HTT gene, alongside genetically matched controls. Furthermore, we provide verified neural stem cells derived from HD patients with >50 CAG repeats, which can be differentiated into striatal neurons exhibiting distinct morphological and functional phenotypes compared to healthy lines. To facilitate complex in vitro modeling, we can also generate cortical excitatory neurons, astrocytes, and microglia from the same HD genetic background, enabling physiologically relevant co-cultures that better replicate disease phenotypes. These patient-derived iPSC-derived neurons, whether striatal or cortical, retain the donor's genetic background, offering high physiological relevance. This allows for detailed modeling of disease progression, assessment of patient-specific responses, and investigation of developmental aspects, ultimately paving the way for personalized medicine approaches in HD.

• Rodent Primary Neurons

To comprehensively support HD research, we offer primary striatal and cortical neurons, the most physiologically relevant cell models. Isolated directly from in vivoHD models, such as R6/2 transgenic mice, or transfected with mutant HTT (mHTT), these neurons provide highly reliable cellular models. Leveraging their native neuronal properties, we perform a range of comprehensive assays, including survival and mHTT assays, tailored to your specific research needs. These primary neuronal cultures are particularly valuable for investigating critical aspects of HD pathogenesis, such as synaptic dysfunction, excitotoxicity, and early neurodegeneration, offering insights that are difficult to obtain with immortalized cell lines.

• Stable Cell Lines

For HD drug discovery, we offer a robust platform utilizing a range of well-characterized cell lines. This includes PC12 and SH-SY5Y cells, engineered to express either the mutant HTT protein with 103 glutamine repeats (Q103) or the normal HTT protein with 25 repeats (Q25), providing valuable tools for comparative studies. Furthermore, we provide the RUES2 cell line, precisely modified at the HTT gene, and an inducible cell line expressing green fluorescent mutant HTT-92Q, enabling powerful investigations into mHTT aggregation, toxicity, and high-throughput small molecule screening. These cell lines offer significant advantages, including high reproducibility, scalability, and cost-effectiveness, making them ideal for developing and validating potential HD therapeutics.

Cell Viability Assay

We assess the detrimental impact of mHTT accumulation on neuronal survival using established methods such as MTT, CCK8, and LDH assays, which are crucial for primary drug screening in HD models.

Fig.1 Neuronal cell viability was measured by MTT assay after the treatment of BDNF for 12 h.^{1, 5}

HTT Aggregation/Lowering Assay

Targeting mHTT aggregation, a key driver of neuronal cell death, is central to HD therapeutics. We employ confocal microscopy to visualize HTT aggregates and utilize RT-qPCR, electrochemiluminescence, and Western blotting to quantify total HTT (tHTT) and mHTT mRNA and protein levels. This allows for the rigorous evaluation of promising disease-modifying approaches, including ASOs, ssRNAs, siRNAs, shRNAs, miRNAs, and splicing modifiers.

Fig.2 Representative images for Htt-25Q and mHtt-94Q containing cells.^{2, 5}

MEA Assay

The Multielectrode Array (MEA) assay provides unparalleled insight into neuronal network dysfunction in HD. With decades of experience, we offer expert assistance in utilizing MEA to record spontaneous action potentials at the single-cell and network levels, enabling the study of HD phenotypes and the assessment of compound efficacy in restoring delayed network formation and diminished spontaneous activity.

Fig.3 Representative image of hPSC-derived neurons on the MEA.^{3, 5}

Mitochondrial Dysfunction Assay

Recognizing the critical role of mitochondrial dysfunction in HD pathogenesis, we utilize the JC-1 fluorescence dye to evaluate mitochondrial membrane potential (MMP). This allows for the precise determination of drug effects on mitochondrial health, distinguishing between energized and depolarized mitochondria, and elucidating mechanisms of neuronal degeneration.

Fig.4 MMP was determined with JC-1 to evaluate the effects of HSF1 in striatal cells.⁴

Creative Biolabs offers a robust platform, encompassing these advanced technologies, to support your HD research. We are dedicated to providing reliable and comprehensive solutions for in vitroand in vivo HD drug screening. Contact us to partner in advancing your HD therapeutic development.

References

1. Hu, Di et al. "Small-molecule suppression of calpastatin degradation reduces neuropathology in models of Huntington's disease." Nat Commun. 2021, 12(1):5305.
2. Li, Li et al. "Real-time imaging of Huntingtin aggregates diverting target search and gene transcription." Elife. 2016, 5:e17056.
3. Kapucu, F.E., Vinogradov, A., Hyvärinen, T. et al. "Comparative microelectrode array data of the functional development of hPSC-derived and rat neuronal networks." Sci Data. 2022, 9:120.
4. Intihar TA et al. "Mitochondrial Dysfunction in Huntington's Disease; Interplay Between HSF1, p53 and PGC-1α Transcription Factors." Front. Cell. Neurosci. 2019, 13:103. Distributed under Open Access License CC BY 4.0 without modification.
5. Distributed under Open Access License CC BY 4.0. The original image was modified.