Fetal microchimeric cells in a fetus-treats-its-mother paradigm do not contribute to dystrophin production in serially parous mdx females

Abstract

Throughout every pregnancy, genetically distinct fetal microchimeric stem/progenitor cells (FMCs) engraft in the mother, persist long after delivery, and may home to damaged maternal tissues. Phenotypically normal fetal lymphoid progenitors have been described to develop in immunodeficient mothers in a fetus-treats-its-mother paradigm. Since stem cells contribute to muscle repair, we assessed this paradigm in the mdx mouse model of Duchenne muscular dystrophy. mdx females were bred serially to either ROSAeGFP males or mdx males to obtain postpartum microchimeras that received either wild-type FMCs or dystrophin-deficient FMCs through serial gestations. To enhance regeneration, notexin was injected into the tibialis anterior of postpartum mice. FMCs were detected by qPCR at a higher frequency in injected compared to noninjected side muscle (P=0.02). However, the number of dystrophin-positive fibers was similar in mothers delivering wild-type compared to mdx pups. In addition, there was no correlation between FMC detection and percentage dystrophin, and no GFP+ve FMCs were identified that expressed dystrophin. In 10/11 animals, GFP+ve FMCs were detected by immunohistochemistry, of which 60% expressed CD45 with 96% outside the basal lamina defining myofiber contours. Finally we confirmed lack of FMC contribution to statellite cells in postpartum mdx females mated with Myf5-LacZ males. We conclude that the FMC contribution to regenerating muscles is insufficient to have a functional impact.

FIG. 1.

mdx breeding strategy and pregnancy history. (A) Experimental group: mdx females mated serially with ROSAeGFP males that harbor a single copy of GFP in the ROSA26 locus. In this breeding strategy, 50% of the progeny and thus trafficked FMCs are labeled by the paternally inherited transgene. Female FMCs that traffic into their mdx mothers are also unaffected by the dystrophin mutation, and thus essentially normal. (B) Control group: mdx females mated serially, but only with mdx males. As a consequence, all the trafficked FMCs are affected by the dystrophin mutation (no normal cells). (C) Pregnancy history: mdx females mated with ROSAeGFP males had at least 2 pregnancies. If litters were not cannibalized, the sex and genotype of the progeny were recorded. M, male; F, female; ?, sex, and genotype of pups were unknown due to their cannibalization; FMC, fetal microchimeric stem/progenitor cells; GFP, green fluorescent protein.

FIG. 2.

More fetal gDNA is specifically detected in mdx TA muscles after treatment with notexin. (A) GFP gDNA is present only in mdx females mated with GFP males. GFP gDNA is selectively found in mdx TA muscles treated with notexin (paired t-test P=0.02). GFP gDNA was also detected in saline-injected TA muscles of some dystrophic mice. (B) The amount of GFP sequence detected in mdx muscles correlates with the number of GFP+ve pups delivered by mdx females (Pearson's r P=0.01, R²=0.6). TA, tibialis anterior.

FIG. 3.

FMCs do not contribute substantially to dystrophin levels in regenerating TA muscles. (A) Examples of dystrophin+ve fibers (red) in mdx TA 7 days after local administration; (a) saline, (b) notexin, (c) or in wild-type TA and (d) rabbit IgG isotype control. White arrows point to revertant clusters. Scale bars represent 50 μM. All images were taken with the Zeiss LSM and Zen 2008 software. (B) Percentage of individual dystrophin+ve fibers relative to the total number of fibers. (C) Total number of dystrophin+ve clusters. The graphs represent pooled data from 2-sectional depths. Color images available online at www.liebertpub.com/scd

FIG. 4.

Levels of dystrophin+ve fibers in regenerating TA muscles cannot be attributed to female progeny. Linear regression analyses comparing dystrophin percentage to the number of female fetuses delivered among those of known gender (A) or percentage of female fetuses delivered (B) showed no correlation. (C) GFP gDNA detection in postpartum mdx females did not be correlate with dystrophin+ve fiber detection by IHC.

FIG. 5.

GFP+ve FMCs detected among maternal muscle fibers are mostly CD45+ve and outside the basal lamina. (A) Intact mononuclear GFP+ve FMCs (green), indicated by white arrows, were found in spaces between muscle fibers and appeared to be part of the inflammatory infiltrate. (B) Total number of FMCs found in 12 sections scanned. Although one false+ve cell was found in a control animal, the data were significant; P=0.005 by one-tailed Mann–Whitney. (C) The presence of GFP+ve FMCs in at least 2 of 3 slides scanned. (D) Examples of GFP+ve FMCs (green) that express surface CD45 (red) (white arrows). (E) Examples of GFP+ve FMCs (green) that are vimentin+ve (red) (white arrows) and vimentin–(yellow arrows). (F-a and F-b) Examples of GFP+ve FMCs (green) detected between fibers outside the basal lamina (red) (yellow arrows). (F-c and F-d) Examples of dystrophin–ve GFP+ve FMCs (green) (yellow arrows). (G) Proportion of FMCs co-labeled with CD45, vimentin, laminin, or dystrophin. All nuclei are stained with DAPI (blue). Scale bars represent 20 μm. All images were captured with the Zeiss Axio Imager M1 fluorescent microscope. Color images available online at www.liebertpub.com/scd