Human Müller glia with stem cell characteristics differentiate into retinal ganglion cell (RGC) precursors in vitro and partially restore RGC function in vivo following transplantation

Abstract

Müller glia with stem cell characteristics have been identified in the adult human eye, and although there is no evidence that they regenerate retina in vivo, they can be induced to grow and differentiate into retinal neurons in vitro. We differentiated human Müller stem cells into retinal ganglion cell (RGC) precursors by stimulation with fibroblast growth factor 2 together with NOTCH inhibition using the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). Differentiation into RGC precursors was confirmed by gene and protein expression analysis, changes in cytosolic [Ca(2+)] in response to neurotransmitters, and green fluorescent protein (GFP) expression by cells transduced with a transcriptional BRN3b-GFP reporter vector. RGC precursors transplanted onto the inner retinal surface of Lister hooded rats depleted of RGCs by N-methyl-d-aspartate aligned onto the host RGC layer at the site of transplantation but did not extend long processes toward the optic nerve. Cells were observed extending processes into the RGC layer and expressing RGC markers in vivo. This migration was observed only when adjuvant anti-inflammatory and matrix degradation therapy was used for transplantation. RGC precursors induced a significant recovery of RGC function in the transplanted eyes as determined by improvement of the negative scotopic threshold response of the electroretinogram (indicative of RGC function). The results suggest that transplanted RGC precursors may be capable of establishing local interneuron synapses and possibly release neurotrophic factors that facilitate recovery of RGC function. These cells constitute a promising source of cells for cell-based therapies to treat retinal degenerative disease caused by RGC dysfunction.

Figure 1.

Notch inhibition promotes differentiation of human Müller stem cells (hMSCs) into retinal ganglion cell precursors in vitro. (A): Western blot showing that decreases in the levels of active NOTCH (ICD) are accompanied by increases in BRN3b protein expression in hMSCs cultured with FGF2 alone or combined with DAPT. (B): hMSCs cultured with FGF2+DAPT showed a decrease in the intensity of staining for the FL and active forms (ICD) of NOTCH as compared with control cells. (C, D): Cells cultured with FGF2+DAPT not only showed enhanced staining for ISL1 and HUD as compared with cells cultured on matrix alone, but also the proportion of cells staining for these markers was significantly increased as shown by histograms (ISL1: *, p = .0003; HUD: **, p = .0044, vs. FGF2 alone). (E): hMSCs cultured with FGF2+DAPT showed a decrease in the number of cells staining for the proliferating antigen Ki67 (***, p = .0048; n = 5). Abbreviations: DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; DMSO, dimethyl sulfoxide; FGF, fibroblast growth factor; FL, full-length; HUD, Hu antigen D protein; ICD, intracellular domain; ISL, insulin gene enhancer protein.

Figure 2.

NOTCH inhibition promotes acquisition of neural morphology by human Müller stem cells (hMSCs) in vitro. (A): Characteristic glial cell morphology of hMSCs cultured in the absence of growth or differentiation factors. (B): hMSCs cultured for 7 days with FGF2 exhibited characteristic cytoplasmic elongations. (C): hMSCs cultured with FGF+DAPT acquired a distinct neural morphology with long neurites and phase bright soma. (D): Histogram shows a significant increase in the number of neurites formed by cells cultured with FGF2 alone (*, p = .03) or FGF2+DAPT (**, p = .0041) compared with untreated (control) cells; n = 3. (E): Histogram shows a significant increase in the length of neurites formed by cells cultured with FGF2 alone (*, p = .02) or FGF2+DAPT (**, p = .0367) compared with untreated (control) cells; n = 3. Abbreviations: DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; DMSO, dimethyl sulfoxide; FGF, fibroblast growth factor.

Figure 3.

BRN3b/GFP reporter-positive cells exhibited retinal ganglion cell (RGC) precursor phenotype in vitro. (A): Colony of human Müller stem cells (hMSCs) transfected with the BRN3b-GFP reporter vector and cultured with fibroblast growth factor 2 (FGF2)+DAPT. (B): Individual hMSCs cultured with FGF2+DAPT for 7 days showing a striking neural morphology with formation of long and ramified neurites. (C): Reverse transcription-polymerase chain reaction analysis of FACS of cells cultured with FGF2+DAPT into GFP+ and GFP− cells showed significantly higher levels of BRN3b mRNA expression in BRN3b/GFP+ cells than in BRN3b/GFP− cells (*, p = .0029; n = 4). (D): hMSCs transfected with the BRN3b-GFP reporter and cultured with FGF2+DAPT, costained for ISL1 (red) and GFP. Histogram shows that BRN3b/GFP+ cells had a significantly higher number of cells expressing nuclear ISL1 (**, p = .0008; n = 4) than BRN3b/GFP− cells. (E): FACS-sorted hMSCs cultured with FGF2+DAPT showed a significantly lower number of Ki67-positive cells in the BRN3b/GFP+ population (***, p < .001) than in the BRN3b/GFP− population. Abbreviations: DAPI, 4′,6′-diamino-2-phenylindole; DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; GFP, green fluorescent protein.

Figure 4.

Retinal ganglion cell (RGC) precursors derived from human Müller stem cells (hMSCs) exhibit neural function in vitro. (A): Heat map photomicrographs of hMSCs cultured with and without FGF2+DAPT for 7 days. After this period of differentiation, cells were loaded with Fura Red dye and stimulated with 2 mM nicotine. Spectrum of yellow (lowest) to red (highest) indicates calcium levels. Cells treated with FGF2+DAPT showed a significant increase in cytoplasmic calcium upon stimulation with nicotine (arrowheads). (B): Kinetics of calcium response showing differences between control (black line) and FGF2+DAPT-treated cells (red line) and its specific inhibition by the nicotinic inhibitor methyl-lycaconitine (100 μM) (blue line). The black arrow indicates the time at which nicotine and the inhibitor were added to the cells. (C): Enlarged sequential images of the cell delineated with dotted lines in FGF2+DAPT-treated cells in (A), illustrating changes in calcium levels upon nicotinic stimulation. (D): Histogram showing the number of responsive control and FGF2+DAPT-treated cells over the total tested in response to nicotine stimulation. Scale bars = 20 μm. Abbreviations: DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; FGF, fibroblast growth factor.

Figure 5.

Localization of retinal ganglion cell (RGC) precursors following transplantation into RGC-depleted retina. (A): Confocal projection of RGC-depleted retina 4 weeks after transplantation with human Müller stem cell-derived RGC precursors expressing the BRN3b-GFP reporter. Transplanted RGC precursors (GFP+) were observed aligning the RGC layer. In several places, GFP+ cells extended neuronal-like processes into the GCL, where they costained for the synaptic protein synaptophysin (red). Side panel shows a magnification of the dotted area. Arrows show colocalization of synaptophysin with BRN3b GFP+ cells (yellow). (B): Section of a transplanted rat retina stained for synaptophysin (red). Upper panel shows DAPI staining of apoptotic host nuclei (indicated by arrowheads) in close proximity to transplanted human cell nuclei, as identified by their large size (open arrows). The lower panel shows the distribution of the GFP-positive cells in relation to the host's cells in the same section. (C): Section of a transplanted rat retina stained for the RGC marker ISL1 (magenta). Lower panel shows a magnification of the dotted area where transplanted BRN3b GFP+ cells can be seen expressing the RGC marker ISL1 (white arrows). (D): Section of a transplanted rat retina stained for NF (red), showing colocalization of NF protein with the BRN3b GFP+ cells (white arrows). Magnification of the dotted area to show detailed colocalization of NF and GFP is presented in the side panel. Scale bars = 20 μm. Abbreviations: DAPI, 4′,6′-diamino-2-phenylindole; GCL, ganglion cell layer; GFP, green fluorescent protein; INL, inner nuclear layer; IPL, inner plexiform layer; NF (RT97), neurofilament protein; ONL, outer nuclear layer; OPL, outer plexiform layer; SYN, synaptophysin.

Figure 6.

Transplantation of retinal ganglion cell (RGC) precursors derived from human Müller stem cells (hMSCs) partially restore the nSTR in RGC-depleted retina. (A): Control (untreated) eyes showed a characteristic scotopic threshold response curve with positive and negative (nSTR, black curve) responses. RGC-depleted eyes without transplantation (NMDA/TA) showed a small positive scotopic threshold response (pSTR) but did not demonstrate a nSTR (blue curve). In contrast, transplanted RGC-depleted eyes showed a small pSTR accompanied by a partial but significant recovery of the nSTR when compared with the nontransplanted eyes (pink curve, p = .0214). (B): Scatter plots comparing the nSTR amplitudes of the controls and transplanted eyes. The amplitude of the nSTR of RGC-depleted eyes transplanted with RGC precursors was significantly lower than that of the control eyes (*, p < .01) but significantly higher than the nSTR observed with nontransplanted RGC-depleted retina (**, p = .0214). Of the 10 eyes included in the transplant group, 8 were found to have good histological transplant survival and integration, whereas 2 (red dots on the graph) did not. Note that these two data points have higher nSTR amplitudes compared with the others. Error bars = SEM. (C): Representative electroretinogram (ERG) responses of control, RGC-depleted eyes without transplantation (NMDA/TA) and transplanted RGC-depleted eyes (NMDA/TA/Transpl). Note the difference in curves at −4 log cd sm⁻², the intensity at which STRs were measured. Abbreviations: NMDA, N-methyl-d-aspartate; nSTR, negative scotopic threshold response; TA, triamcinolone.