Dermal Fibroblast Lines from a Patient with Huntington’s Disease as a Promising Model for Studying the Pathogenesis of the Disease: Production and Characterization

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Abstract

Huntington’s disease (HD) is an inherited, incurable disease caused by a mutation and expansion of CAG repeats in the HTT gene encoding the huntingtin protein (mHTT). Despite numerous studies conducted on various cellular and animal models, specific mechanisms explaining the biological role of mHTT and its toxicity to striatal neurons have not yet been established, and effective therapy for patients has not been developed. We obtained and characterized a new line of dermal fibroblasts HDDF (Huntington Disease Dermal Fibroblasts) from a patient with a confirmed diagnosis of HD. The growth characteristics of the HDDF line, staining for canonical markers, karyotyping and cell phenotyping were performed. Direct differentiation of fibroblasts from the resulting line into induced striatal neurons was carried out. The new fibroblast line can be used as a cell model to study the biological role of mHTT and various manifestations of HD pathogenesis both in fibroblasts themselves and in induced neuronal cells obtained using reprogramming methods.

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About the authors

N. Kraskovskaya

Institute of Cytology of the Russian Academy of Sciences

Author for correspondence.
Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

A. Koltsova

Institute of Cytology of the Russian Academy of Sciences

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

P. Parfenova

Institute of Cytology of the Russian Academy of Sciences

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

A. Shatrova

Institute of Cytology of the Russian Academy of Sciences

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

N. Yartseva

Institute of Cytology of the Russian Academy of Sciences

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

V. Nazarov

Pavlov First Saint Petersburg State Medical University

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

E. Devyatkina

Pavlov First Saint Petersburg State Medical University

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

M. Khotin

Institute of Cytology of the Russian Academy of Sciences

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

N. Mikhailova

Institute of Cytology of the Russian Academy of Sciences

Email: ninakraskovskaya@gmail.com
Russian Federation, Saint Petersburg

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2. Fig. 1. General view of the obtained fibroblast line. a - Lifelike micrograph of the biopsy fragment and primary fibroblasts. Light microscopy. Scale of 200 μm. b and c - Lifelong micrographs of dermal fibroblast lineage at the 7th passage at 10× and 20× magnification, respectively. Light microscopy. Scale 100 μm

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3. Fig. 2. Karyotype of HDDF cell line with normal chromosome number 46 XX, without structural chromosome rearrangements

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4. Fig. 3. Growth characteristics of the HDDF line. Graph illustrating the growth curve of the line at the 7th, 20th, 30th and 40th passage

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5. Fig. 4. Replicative senescence analysis of the HDDF line at the 7th, 20th, 30th and 40th passages. a, b, c, d - Microphotographs illustrating β-galactosidase activity in cells at the 7th, 20th, 30th and 40th passages, respectively. e - Histogram illustrating the results obtained. Data are presented as mean ± error of the mean, **** p < 0.001 according to one-way ANOVA analysis with Dunn's a posteriori test. At least 1000 cells were analysed at each passage

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6. Fig. 5. Phenotyping of HDDF cells by flow cytofluorimetry (a-h)

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7. Fig. 6. Immunofluorescence staining of cells for fibroblast markers. a - Staining with antibodies to vimentin. b - Staining with antibodies to α-actinin. c - Staining with antibodies to nestin. Nuclei were visualised with DAPI dye. Confocal microscopy, ×40. Scale 50 µm

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8. Fig. 7. Microphotographs of induced striatum neurons derived from HDDF fibroblasts. a - Immunofluorescence staining with antibodies to MAP2 marker. b - Immunofluorescence staining with antibodies to TUJ-1 marker. c - Immunofluorescence staining with antibodies to GABA marker. d - Immunofluorescence staining with antibodies to DARPP-32 marker. Confocal microscopy, ×60. Scale bar 50 µm

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9. Fig. 8. Visualisation of mHTT in fibroblasts and induced neurons of the HDDF line. a - Immunofluorescence staining of fibroblasts with antibodies to mHTT (clone mEM48). Confocal microscopy, ×60. Scale bar 50 μm. b - Immunofluorescence staining of induced striatum neurons derived from fibroblasts of the HDDF line with mEM48 antibodies (secondary antibodies conjugated to Alexa 488 fluorophore) and staining of nuclei with DAPI dye. Confocal microscopy, ×60. Scale bar 20 μm

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