Healthy, mtDNA-mutation free mesoangioblasts from mtDNA patients qualify for autologous therapy

Florence van Tienen, Ruby Zelissen, Erika Timmer, Marike van Gisbergen, Patrick Lindsey, Mattia Quattrocelli, Maurilio Sampaolesi, Elvira Mulder-den Hartog, Irenaeus de Coo, Hubert Smeets, Florence van Tienen, Ruby Zelissen, Erika Timmer, Marike van Gisbergen, Patrick Lindsey, Mattia Quattrocelli, Maurilio Sampaolesi, Elvira Mulder-den Hartog, Irenaeus de Coo, Hubert Smeets

Abstract

Background: Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy.

Methods: We assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity.

Results: This study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity.

Conclusions: Our data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function.

Keywords: Mesoangioblasts; Muscle regeneration; mtDNA mutation.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
mtDNA mutation load per mtDNA point-mutation in single mesoangioblasts. The mtDNA mutation load in single mesoangioblasts in carriers from 5 different mtDNA point-mutations: a m.3243A>G, b m.3271 T>C, c m.3291 T>C, d m.8363G>A, and e m.11778G>A. Each gray dot represents the mtDNA mutation load in a single mesoangioblast (> 15 per person were analyzed). Asterisk indicates mean mtDNA mutation load in skeletal muscle. Dotted black line indicates median mtDNA mutation load analyzed in single mesoangioblasts
Fig. 2
Fig. 2
Semi-quantitative analysis of large-scale mtDNA deletions in mesoangioblasts and skeletal muscle. The 16.5 kb mtDNA was PCR amplified and analyzed on a 0.7% agarosegel. M, mesoangioblasts; S, skeletal muscle
Fig. 3
Fig. 3
Myogenic potency of mesoangioblasts from mtDNA carriers. The myogenic potential was quantified following 10 days of differentiation in 2% horse serum containing medium and quantification of the myogenic fusion index, namely the number of nuclei (DAPI) in myosin-positive (MF20) muscle fibers per total number of nuclei per field. a Example image of 10 days differentiated MABs following MF20 immunostaining of myotubes (green) and DAPI nuclear stain (blue). b Interaction between age at biopsy and the mesoangioblast mtDNA mutation load (irrespective of type of mtDNA mutation) on the spontaneous myogenic potential of mesoangioblasts, using the best linear regression model obtained and described by E(Myogenic fusion index) = 0.146 − 0.00155 × age − 0.0394 × ln(MABs) + 0.00056 × age × ln(MABs)
Fig. 4
Fig. 4
Mesoangioblast mtDNA copy number. The mean mtDNA copy number per cell line was determined by qPCR analysis of mtDNA D-loop and nuclear B2M. Per sample, the mean mtDNA copy number ± S.E.M. is shown. The solid line represents the mean mtDNA copy number calculated from all samples (n = 27), and dashed lines indicate mean ± 2 S.D.
Fig. 5
Fig. 5
Mitochondrial function. Mitochondrial respiration in MABs of tRNAleu mutation carriers was determined by measuring the oxygen consumption rate (OCR) in a Seahorse XF96 analyzer during treatment with oligomycin (oligo), FCCP and Antimycin/Rotenone (ant/rot), and corrected for cell number. Per cell line, 8 replicates were included; mean OCR ± S.D. is shown in figure

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