The Benefits and Hazards of Intravitreal Mesenchymal Stem Cell (MSC) Based-Therapies in the Experimental Ischemic Optic Neuropathy

Abstract

Mesenchymal stem cell (MSC) therapy has been investigated intensively for many years. However, there is a potential risk related to MSC applications in various cell niches.

Methods: The safety of intravitreal MSC application and the efficacy of MSC-derived conditioned medium (MDCM) were evaluated in the normal eye and the diseased eye, respectively. For safety evaluation, the fundus morphology, visual function, retinal function, and histological changes of the retina were examined. For efficacy evaluation, the MDCM was intravitreally administrated in a rodent model of anterior ischemic optic neuropathy (rAION). The visual function, retinal ganglion cell (RGC) density, and neuroinflammation were evaluated at day 28 post-optic nerve (ON) infarct.

Results: The fundus imaging showed that MSC transplantation induced retinal distortion and venous congestion. The visual function, retinal function, and RGC density were significantly decreased in MSC-treated eyes. MSC transplantation induced astrogliosis, microgliosis, and macrophage infiltration in the retina due to an increase in the HLA-DR-positive MSC proportion in vitreous. Treatment with the MDCM preserved the visual function and RGC density in rAION via inhibition of macrophage infiltration and RGC apoptosis.

Conclusions: The vitreous induced the HLA-DR expression in the MSCs to cause retinal inflammation and retina injury. However, the MDCM provided the neuroprotective effects in rAION.

Keywords: HLA-DR expression; MSC-derived conditioned medium; mesenchymal stem cell; rodent model of anterior ischemic optic neuropathy.

Figure 1

The intravitreal delivery of human Wharton’s jelly mesenchymal stem cells (hWJMSCs) in rat retina four weeks after transplantation. (A) Ocular color fundus images in the PBS-treated group and the hWJMSC-treated group. The black arrowhead indicates retinal venous congestion. Red fluorescence shows the DiI-labeled hWJMSCs in the vitreous cavity. Digital magnification (800%) (B) Hematoxylin and eosin staining of retinal sections. The hWJMSC transplantation resulted in retinal structural deformity. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Scale bar 200 µm. (C) Immunohistochemistry of human mitochondria staining and DiI-labeled cells in the PBS-treated group and the hWJMSC-treated group. Scale bar 100 µm. DAPI, 4, 6-diamidino-2-phenylindole; h-mito, human-mitochondria; DiI: 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate.

Figure 2

Evaluation of visual function and retinal function through flash visual-evoked potential (FVEP) and electroretinogram (ERG) recordings. (A) Bar chart showing the P1-N2 amplitudes in the PBS-treated group and in the hWJMSC group. A significant decrease of P1-N2 amplitude was found in the hWJMSC-treated group as compared to that in the PBS-treated group (p < 0.05, n = 12). Data are expressed as mean ± SD. P1, P1 the first positive peak; N2, the second negative peak. (B) Recordings of scotopic and (C) photopic ERG analysis revealed the amplitudes of a- and b-wave in the PBS-treated group and the hWJMSC-treated group. Both visual electrophysiology data showed that the amplitudes of a- and b-waves were significantly reduced by hWJMSC treatment compared to the PBS treatment. Data are expressed as mean ± SD.* p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Analysis of the density of Brn3a-positive cell in the PBS-treated group and the hWJMSC-treated group. (A) Representative images of Brn3a-positive cells in the GCL. (B) Quantification of Brn3a-positive cell density in the PBS-treated group and the hWJMSC group. *** p < 0.0001. Scale bar: 100 µm. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer; ONL: outer nuclear layer; DAPI, 4, 6-diamidino-2-phenylindole; Brn3a, brain-specific homeobox/POU domain protein 3A; DiI: 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate.

Figure 4

Immunoreactivity of Iba1, ED1, and GFAP in the retina after hWJMSCs transplantation. Representative image of microglia (Iba1+), macrophage (CD68+), and astrocyte (GFAP+) in each group. The confocal image showed the retinal inflammation 28 days after hWJMSCs transplantation. ED1-positive cells infiltrated into the subretinal space (arrowheads). Scale bar: 100 µm. DAPI: 4, 6-diamidino-2-phenylindole; Iba1: ionized calcium binding adaptor molecule 1; ED1: Macrosialin; GFAP: Glial fibrillary acidic protein; DiI: 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer.

Figure 5

The HLA-DR positive hWJMSC in the vitreous cavity four weeks after transplantation. Representative image of HLA-DR staining in the vitreous cavity. The HLA-DR-positive cells and the nuclei were labeled in green and blue colors, respectively. DiI was used to label the transplanted hWJMSC in red color. Scale bar: 20 µm. DAPI, 4, 6-diamidino-2-phenylindole; DiI: 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate; HLA-DR: MHC class II antigen.

Figure 6

FVEP profiles at day 28 post-rAION (rodent model of anterior ischemic optic neuropathy) induction. (A) Representative FVEP wavelet in each group. (B) Treatment with CM induced the P1-N2 amplitude by 1.97-fold compared to treatment with SFM (* p < 0.05, n = 12 per group). Data are expressed as mean ± SD. SFM: serum-free medium; CM: conditioned medium. P1, P1 the first positive peak; N2, the second negative peak; ns, not significant

Figure 7

The RGC density and the number of apoptotic RGCs in the 4th week after rAION induction (A) Representative images of RGCs (Brn3a-positive cell) in red and TUNEL-positive cells in green. (B) Quantification of the RGC density and the number of TUNEL-positive cells. Treatment with CM significantly preserved the RGC density after ON infarct. The apoptotic RGCs were significantly reduced after CM treatment. * p < 0.05; *** p < 0.001. Scale bar: 40 µm. DAPI, 4, 6-diamidino-2-phenylindole; Brn3a, brain-specific homeobox/POU domain protein 3A; TINEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer; ONL: outer nuclear layer.

Figure 8

Microglia activation and macrophage infiltration in the optic nerve after ON infarct. (A) representative image of Iba1 and ED1 staining in longitudinal sections of the ONs. The Iba1-positive cells, the ED1-positive cells, and the nuclei are marked in green, red, and blue colors, respectively; Treatment with CM effectively reduced the size of the microglia cell (Iba1-positive cell) in contrast to treatment with SFM four weeks after ON infarct. (B) Quantification of Iba1+ and ED1+ cells per high-power field (HPF). Data are expressed as mean ± SD (n = 6 per group). The decreasing number of Iba1+ cells was significantly different in the CM-treated group as compared to in the SFM-treated group (n = 6 per group). The number of macrophages (ED1-positive cells) in the CM-treated group was lower than that in the SFM-treated group (n = 6 per group). Scale bar: 100 µm. * p < 0.05. ** p < 0.01. Iba1: ionized calcium binding adaptor molecule 1; ED1: Macrosialin.

Figure 9

Study design to evaluate the safety and the efficacy of intravitreal MSC-based therapies. The safety of intravitreal injection of hWJMSC was investigated in the normal Wistar rat by using fundus photography, FVEP, ERG, and IHC analysis. The therapeutic effects of intravitreal injection of the hWJMSC-derived CM were evaluated in the rAION model by using FVEP, Brn3a staining, TUNEL assay, ED1 staining, Iba1 staining, and GFAP staining.