Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS - PubMed (original) (raw)
. 2014 Jan 14;111(2):829-32.
doi: 10.1073/pnas.1314085111. Epub 2013 Dec 30.
Laura Ferraiuolo, Carlos J Miranda, Shibi Likhite, Sohyun McElroy, Samantha Renusch, Dara Ditsworth, Clotilde Lagier-Tourenne, Richard A Smith, John Ravits, Arthur H Burghes, Pamela J Shaw, Don W Cleveland, Stephen J Kolb, Brian K Kaspar
Affiliations
- PMID: 24379375
- PMCID: PMC3896192
- DOI: 10.1073/pnas.1314085111
Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS
Kathrin Meyer et al. Proc Natl Acad Sci U S A. 2014.
Abstract
Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.
Keywords: neurodegeneration; neurotoxicity; reprogramming.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Direct conversion of human skin fibroblasts to tripotent iNPCs. (A) Schematic of the conversion process from fibroblasts to induced neuronal progenitor cells (iNPCs). Fibroblasts were transduced with retroviral vectors containing four reprogramming factors (Sox2, KLF4, Oct3/4, c-Myc). (B and C) Within 6–10 d, cells underwent marked morphological changes from a fibroblastic spindle like shape (B) to a sphere-like form commonly seen with NPCs (C). (D and E) Immunofluorescence of cultures at day 12 reveals expression of the NPC markers Pax6 and Nestin, as shown in red. DAPI staining (blue) was used to visualize nuclei. (F) iNPCs can form and grow as neurospheres when plated in uncoated dishes. (G) RT-PCR analysis demonstrates a strong up-regulation of the prototypic NPC markers NCAN and NKX2-2 in iNPCs. β-Actin was used as loading control. (H–J) iNPCs are tripotent and upon differentiation they can give rise to oligodendrocytes (H), neurons (I), and astrocytes (J). (Scale bars: black, 100 µm; white, 50 μm.) Fibro, fibroblast.
Fig. 2.
I-astrocytes express prototypic astrocyte markers. (A) Immunofluorescence analysis reveals strong up-regulation of astrocytic markers such as Vimentin, CD44, S100β, and GFAP in i-astrocytes compared with the initial fibroblasts. DAPI (blue) was used to visualize nuclei. (B) RT-PCR analysis revealed expression of IGFB3 in fibroblasts, iNPCs, and i-astrocytes, whereas expression of S100β and Aqp4 was detected in iNPCs and i-astrocytes or i-astrocytes only, respectively. (Scale bar: 100 μm.) Fibro, fibroblast. i-Astro, i-astrocytes.
Fig. 3.
I-astrocytes from fALS and sALS patients display toxicity toward MNs. (A) Representative images after 96 h of coculture of HB9-GFP expressing MNs (shown in black) with astrocytes from spinal cord (sc) or skin of ALS patients and controls. A marked loss of MN viability was observed in the presence of ALS astrocytes irrespective of their origin (spinal cord or skin). (B) Relative percentage of MN survival after 96 h of coculture with ALS astrocytes derived from spinal cord or skin and their respective controls. ***P < 0.001; ****P < 0.0001 [compared with the average taken from of all converted control lines (HDFA, 8620, 155, 170)]. Error bars represent SEM. Quantification was performed in triplicate wells of a 96-well plate, and data are representative of n = 5.
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