Tracking of autologous adipose tissue-derived mesenchymal stromal cells with in vivo magnetic resonance imaging and histology after intralesional treatment of artificial equine tendon lesions--a pilot study

Abstract

Background: Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine tendinopathies. Up to now, knowledge about the fate of autologous AT-MSCs after intralesional injection into equine superficial digital flexor tendons (SDFTs) is very limited. The purpose of this study was to monitor the presence of intralesionally injected autologous AT-MSCs labelled with superparamagnetic iron oxide (SPIO) nanoparticles and green fluorescent protein (GFP) over a staggered period of 3 to 9 weeks with standing magnetic resonance imaging (MRI) and histology.

Methods: Four adult warmblood horses received a unilateral injection of 10 × 10(6) autologous AT-MSCs into surgically created front-limb SDFT lesions. Administered AT-MSCs expressed lentivirally transduced reporter genes for GFP and were co-labelled with SPIO particles in three horses. The presence of AT-MSCs in SDFTs was evaluated by repeated examinations with standing low-field MRI in two horses and post-mortem in all horses with Prussian blue staining, fluorescence microscopy and with immunofluorescence and immunohistochemistry using anti-GFP antibodies at 3, 5, 7 and 9 weeks after treatment.

Results: AT-MSCs labelled with SPIO particles were detectable in treated SDFTs during each MRI in T2*- and T1-weighted sequences until the end of the observation period. Post-mortem examinations revealed that all treated tendons contained high numbers of SPIO- and GFP-labelled cells.

Conclusions: Standing low-field MRI has the potential to track SPIO-labelled AT-MSCs successfully. Histology, fluorescence microscopy, immunofluorescence and immunohistochemistry are efficient tools to detect labelled AT-MSCs after intralesional injection into surgically created equine SDFT lesions. Intralesional injection of 10 × 10(6) AT-MSCs leads to the presence of high numbers of AT-MSCs in and around surgically created tendon lesions for up to 9 weeks. Integration of injected AT-MSCs into healing tendon tissue is an essential pathway after intralesional administration. Injection techniques have to be chosen deliberately to avoid reflux of the cell substrate injected. In vivo low-field MRI may be used as a non-invasive tool to monitor homing and engraftment of AT-MSCs in horses with tendinopathy of the SDFT.

Fig. 1

Photomicrographs of cultured adipose-derived mesenchymal stromal cells from horses 1 to 4 after transfection with lentiviral green fluorescent protein (GFP) construct. Bright field (left), fluorescence (middle) and merged (right) images are shown. Approximately 70–90 % of the cells were positive for GFP in their cytoplasm during passage 1. Magnification: 10×. Scale bar: 100 μm

Fig. 2

Flow cytometric analysis of cultured AT-MSCs from horses 1 to 4 after transfection with lentiviral green fluorescent protein (GFP) construct. From left to right are (a) scatter plots and (b) histograms, including percentages showing the distribution of GFP-expressing cells in a non-expressing control sample (ctrl-no GFP) and in samples from horse 2 (I-VP1; passage 0) and horses 1, 3 and 4 (I-VP3, I-VP4 and I-VP2; passage 2) after a single analysis. Green indicates GFP-labelled AT-MSCs, and blue indicates non-labelled AT-MSCs. AT-MSC adipose tissue-derived mesenchymal stromal cell, FSC-A forward scatter A, GFP green fluorescent protein, SSC-A side scatter A

Fig. 3

Photomicrographs of GFP-labelled equine AT-MSCs from horse 1 in cell culture. In the upper row, GFP-fluorescence, anti-GFP immunohistochemistry (DAB) and merged images are shown. In the lower row, GFP-fluorescence, anti-GFP immunofluorescence (Alexa 546) and merged images of AT-MSCs (passage 6) are shown. Magnification: 20×. AT-MSC adipose tissue-derived mesenchymal stromal cell, DAB diaminobenzidine tetrachloride, DAPI 4′,6-diamidino-2-phenylindole, GFP green fluorescent protein

Fig. 4

Photomicrographs of superficial digital flexor tendon lesions 3, 5, 7 and 9 weeks after transplantation of GFP-labelled autologous adipose tissue-derived mesenchymal stromal cells. Bright field, DAPI nuclear stain and GFP-fluorescence and merged images taken from horses 1–4 are shown. Transplanted cells were positive in the cytoplasm for GFP. Magnification: 20×. DAPI 4′,6-diamidino-2-phenylindole, GFP green fluorescent protein

Fig. 5

Photomicrographs of a superficial digital flexor tendon lesion 3 weeks after transplantation of GFP-labelled autologous adipose tissue-derived mesenchymal stromal cells. In the upper row, bright field, GFP-fluorescence, anti-GFP immunohistochemistry (DAB) and merged (arrows: positive cells) images are shown. In the lower row, DAPI nuclear stain, GFP-fluorescence, anti-GFP immunofluorescence (Alexa 546) and merged images (horse 1) are shown. Approximately 50 % of the cells were positive for GFP in their cytoplasm. Magnification: 20×. DAB diaminobenzidine tetrachloride, DAPI 4′,6-diamidino-2-phenylindole, GFP green fluorescent protein

Fig. 6

Photomicrographs of surgically created superficial digital flexor tendon lesions treated with autologous AT-MSCs and stained with Prussian blue. a, b, d Lesions treated with SPIO-labelled AT-MSCs (horses 1, 2 and 4). c Lesion treated with non-SPIO-labelled AT-MSCs (horse 3). Magnifications: 10× (a-d) and 20× (A/1-D/1). AT-MSC adipose tissue-derived mesenchymal stromal cell, SPIO superparamagnetic iron oxide

Fig. 7

Transverse magnetic resonance imaging images of SDFT lesions treated with 10 × 10⁶ SPIO-labelled AT-MSCs or saline. a, c A focal, moderately demarcated hypointense area is evident in and lateral to the AT-MSC-treated SDFT lesion, area indicated by arrowheads in a. b, d Signal intensity of the saline treated lesion is increased in the contralateral SDFT, area indicated by asterisk in b. a, b: T2*-weighted sequence, 47 days after injection. c, d: T1-weighted sequence, 62 days after injection; horse 4. AT-MSC adipose tissue-derived mesenchymal stromal cell, SDFT superficial digital flexor tendon, SPIO superparamagnetic iron oxide