Gdf 15

Blood samples from KD subjects were collected at convalescent time points (median: 14.5 years; range: 0.9–55.0 years post-KD onset). Blood was collected and separated immediately by centrifugation and stored at −80 °C. We measured EDTA plasma levels of calprotectin, Gal-3, sST2, GDF-15, and serum levels of PIPC by ELISA according to the manufacturer’s instructions: calprotectin, Gal-3, GDF-15: R & D Systems, Minneapolis, MN, USA, sST2: Critical Diagnostics, San Diego, CA, USA and PIPC: Quidel, San Diego, CA, USA.

The serum samples used in this study were collected for analysis from 2012 to 2014. All samples were stored at –80°C until analysis. We measured the GDF‐15 (R&D Systems, Minneapolis, USA) and FGF‐21 (FGF‐21; BioVendor, Brno, Czech Republic) concentrations in duplicate samples by enzyme‐linked immunorbent assay (ELISA). The samples for which the concentration of GDF‐15 or FGF‐21 was higher than the range of detection were diluted 10‐fold with dilution buffer and reanalysed. The value from each assay was compared with reference GDF‐15 and FGF‐21 concentrations. We also measured the levels of conventional biomarkers, such as lactate, pyruvate, the lactate/pyruvate (L/P) ratio, and creatine kinase (CK). In addition, we recorded the height, body weight, BMI, and blood sugar levels. The results of each assay were compared with a linear calibration curve. All assays were performed by a trained scientist (S.Y. and R.S.).
The enrolled MD patients were clinically evaluated for disease severity using two rating systems: the Japanese Mitochondrial Disease Rating Scale (JMDRS)9 and the Newcastle Mitochondrial Disease Rating Scale (NMDAS).10 All scores were also evaluated with respect to the levels of GDF‐15 or FGF‐21 using correlation coefficients.

Neurosphere cultures: Following mechanical dissociation, cells were plated at a density of 10⁵ cells/ml in 24 well plates in culture medium consisting of ice cold Euromed-N basal serum free culture medium, Penicillin-Streptomycin (100 U/ml), glutamine (2 mM) and 2% B27 (Gibco). Human recombinant EGF and FGF-2 (Peprotech) were added to the culture medium at a concentration of 20 ng/ml and 10 ng/ml, respectively with or without GDF15 (10 ng/ml, R&D). Cells were fed with 1/2 the volume of fresh culture medium every 4 d precursors were allowed to proliferate in suspension for 7 days, before mechanical dissociation and counting.
Clonal cultures: Using FACS automated cell deposition one EGFR^high and 10 EGFR^low cells were plated at a density of one cell per well in 96 well/plates in culture medium supplemented with EGF and FGF-2. Clones were scored after 7 d.
Secondary clone formation: Single spheres from clonal cultures were mechanically dissociated, and replated in 96 well plates in EGF and FGF-2 supplemented culture medium at a density of 10³ cells per well. Secondary neurospheres were scored after 7 d.

Dissociated cells were plated in culture medium consisting of Euromed-N basal medium (Euroclone) and 2% B27 (Gibco). Human recombinant EGF (20 ng/ml), FGF2 (10 ng/ml) (Peprotech) and GDF15 (10 ng/ml, R&D) were added as indicated in the text.