Regenerative Surgery of the Corneal Stroma for Advanced Keratoconus: 1-Year Outcomes.

PURPOSE: This study evaluated 1-year safety and efficacy outcomes of corneal stroma cell therapy. Therapy consisted of implanting autologous adipose-derived adult stem cells (ADASc) with or without sheets of decellularized donor human corneal stroma within the stroma of patients with advanced keratoconus. DESIGN: This was a prospective interventional non-randomized series of cases. METHODS: Fourteen consecutive patients were selected and divided into 3 experimental groups. Group A patients underwent implantation of autologous ADASc alone (3 × 106 cells/1 mL) (n = 5). Group B patients received decellularized donor 120-µm-thick corneal stroma lamina alone (n = 5). Group C patients had implantation of recellularized donor lamina with 1 × 106 autologous ADASc plus another 1 × 106 cells/1 mL at the time of the surgery (n = 4). Autologous ADASc were obtained by elective liposuction. Implantation was performed in the corneal stroma through a femtosecond-assisted 9.5-mm diameter lamellar dissection with the patient under topical anesthesia. Twelve months of follow-up data are presented. RESULTS: No complications were observed during the 1-year follow-up, and full corneal transparency was recovered within 3 months in all patients. No patient lost lines of visual acuity. Corrected distance visual acuity improved 0.231, 0.264, and 0.094 Snellen lines in groups 1, 2, and 3, respectively. In group 1, refractive parameters showed an overall stability, whereas in groups 2 and 3, sphere improved 2.35 diopter (D) and 0.625 D, respectively. Anterior keratometry remained stable (group 1) and improved in groups 2 and 3 (mean improvement of 2D). Corneal aberrometry improved significantly. In optical coherence tomography scans, corneal thickness showed a mean improvement of 14.5 µm (group 1) and 116.4 µm (groups 2 and 3) in the central thickness, and new collagen production was observed at the surgical plane (group 1). Confocal biomicroscopy confirmed the host recellularization of the implanted laminas. CONCLUSIONS: Intrastromal implantation of autologous ADASc and decellularized human corneal stroma did not show complications at 1 year of follow-up and were moderately effective for the treatment of advanced keratoconus. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Inhibition of the RhoGTPase Cdc42 by ML141 enhances hepatocyte differentiation from human adipose-derived mesenchymal stem cells via the Wnt5a/PI3K/miR-122 pathway: impact of the age of the donor.

BACKGROUND: Human adipose-derived mesenchymal stem cells (hADSCs) are promising cells that may promote hepatocyte differentiation (Hep-Dif) and improve liver function, but the involvement of Cdc42, a key small RhoGTPase which plays a crucial role in aging, is still not well established. We hypothesized that the inhibition of Cdc42 may rescue the hepatogenic potential of hADSCs derived from aged donors. METHODS: hADSCs isolated from 61 women of different ages were cultured for evaluation of the proliferation of cells, adherence, apoptosis, immunomodulation, immunophenotyping, multipotency, gene expression, and cell function during Hep-Dif. Inhibition of Cdc42 by ML141 was realized during two phases: initiation (days -2 to 14 (D-2/14)) from undifferentiated to hepatoblast-like cells, or maturation (days 14 to 28 (D14/28)) from undifferentiated to hepatocyte-like cells. Mechanistic insights of the Wnt(s)/MAPK/PI3K/miR-122 pathways were studied. RESULTS: Cdc42 activity in undifferentiated hADSCs showed an age-dependent significant increase in Cdc42-GTP correlated to a decrease in Cdc42GAP; the low potentials of cell proliferation, doubling, adherence, and immunomodulatory ability (proinflammatory over anti-inflammatory) contrary to the apoptotic index of the aged group were significantly reversed by ML141. Aged donor cells showed a decreased potential for Hep-Dif which was rescued by ML141 treatment, giving rise to mature and functional hepatocyte-like cells as assessed by hepatic gene expression, cytochrome activity, urea and albumin production, low-density lipoprotein (LDL) uptake, and glycogen storage. ML141-induced Hep-Dif showed an improvement in mesenchymal-epithelial transition, a switch from Wtn-3a/ß-catenin to Wnt5a signaling, involvement of PI3K/PKB but not the MAPK (ERK/JNK/p38) pathway, induction of miR-122 expression, reinforcing the exosomes release and the production of albumin, and epigenetic changes. Inhibition of PI3K and miR-122 abolished completely the effects of ML141 indicating that inhibition of Cdc42 promotes the Hep-Dif through a Wnt5a/PI3K/miR-122/HNF4α/albumin/E-cadherin-positive action. The ML141(D-2/14) protocol had more pronounced effects when compared with ML141(D14/28); inhibition of DNA methylation in combination with ML141(D-2/14) showed more efficacy in rescuing the Hep-Dif of aged hADSCs. In addition to Hep-Dif, the multipotency of aged hADSC-treated ML141 was observed by rescuing the adipocyte and neural differentiation by inducing PPARγ/FABP4 and NeuN/O4 but inhibiting Pref-1 and GFAP, respectively. CONCLUSION: ML141 has the potential to reverse the age-related aberrations in aged stem cells and promotes their hepatogenic differentiation. Selective inhibition of Cdc42 could be a potential target of drug therapy for aging and may give new insights on the improvement of Hep-Dif.

Essential role of ATP6AP2 enrichment in caveolae/lipid raft microdomains for the induction of neuronal differentiation of stem cells.

BACKGROUND: The subcellular distribution of prorenin receptor and adaptor protein ATP6AP2 may affect neurogenesis. In this study, we hypothesized that ATP6AP2 expression and subcellular relocalization from caveolae/lipid raft microdomains (CLR-Ms) to intracellular sites may correlate with neuronal differentiation (Neu-Dif) of adipose-derived mesenchymal stem cells (ADSCs). METHODS: Human ADSCs isolated from 24 healthy donors and 24 patients with neurological disorders (ND) were cultured and induced for Neu-Dif. The mechanism of action of ATP6AP2 and the impact of its localization within the plasma membrane (particularly CLR-Ms) and intracellular sites on several pathways (mitogen-activated protein kinase, Wnt(s) signaling and others) and intracellular calcium and exosome release were evaluated. The impact of CLR-Ms on ATP6AP2 or vice versa was determined by pharmacological disruption of CLR-Ms or siATP6AP2 assays. RESULTS: In patients with ND, loss of ATP6AP2 from CLR-Ms correlated with an inhibition of Neu-Dif and signaling. However, its relocalization in CLR-Ms was positively correlated to induction of Neu-Dif in healthy subjects. An apparent switch from canonical to noncanonical Wnt signaling as well as from caveolin to flotillin occurs concurrently with the increases of ATP6AP2 expression during neurogenesis. Stimulation by renin activates ERK/JNK/CREB/c-Jun but failed to induce ß-catenin. Wnt5a enhanced the renin-induced JNK responsiveness. Gα proteins crosslink ATP6AP2 to caveolin where a switch from Gαi to Gαq is necessary for Neu-Dif. In ATP6AP2-enriched CLR-Ms, the release of exosomes was induced dependently from the intracellular Ca2+ and Gαq. Pharmacological disruption of CLR-M formation/stability impairs both ATP6AP2 localization and Neu-Dif in addition to reducing exosome release, indicating an essential role of ATP6AP2 enrichment in CLR-Ms for the induction of Neu-Dif. The mechanism is dependent on CLR-M dynamics, particularly the membrane fluidity. Knockdown of ATP6AP2 inhibited Neu-Dif but increased astrocytic-Dif, depleted ATP6AP2/flotillin/Gαq but accumulated caveolin/Gαi in CLR-Ms, and blocked the activation of JNK/ERK/c-Jun/CREB/exosome release. siATP6AP2 cells treated with sphingomyelinase/methyl-ß-cyclodextrin reversed the levels of caveolin/flotillin in CLR-Ms but did not induce Neu-Dif, indicating the crucial relocalization of ATP6AP2 in CLR-Ms for neurogenesis. Treatment of ND-derived cells with nSMase showed reversibility in ATP6AP2 abundance in CLR-Ms and enhanced Neu-Dif. CONCLUSIONS: This study gives evidence of the determinant role of CLR-M ATP6AP2 localization for neuronal and oligodendrocyte differentiation involving mechanisms of switches from Gαi/caveolin/canonical to Gαq/flotillin/PCP, the ERK/JNK pathway and Ca2+-dependent release of exosomes and as a potential target of drug therapy for neurodegenerative disorders.

Corneal Stroma Enhancement With Decellularized Stromal Laminas With or Without Stem Cell Recellularization for Advanced Keratoconus.

PURPOSE: This phase 1 study seeks to preliminarily evaluate the safety and efficacy of decellularized human corneal stromal lamina transplantation with or without autologous adipose-derived adult stem cell recellularization within the corneal stroma of patients with advanced keratoconus. DESIGN: Phase 1 clinical trial. METHODS: Femtosecond-assisted 120-µm thickness and 9-mm diameter laminas were obtained from the anterior stroma of human donor corneas and decellularized with a sodium dodecyl sulfate solution. Autologous adipose-derived adult stem cells were obtained by elective liposuction and cultured onto both sides of the lamina. Five patients received the decellularized lamina alone and 4 patients the recellularized lamina into a femtosecond-assisted 9.5-mm diameter lamellar pocket under topical anesthesia. The total duration of follow-up was 6 months. RESULTS: No case showed clinical haze or scarring by month 3. Six months after surgery, patients showed a general improvement of all visual parameters, with a mean unaided visual acuity from 0.109 to 0.232 (P = .05) and corrected distance visual acuity from 0.22 to 0.356 (P = .068). Refractive sphere improved in all patients (from -4.55 diopters [D] to -2.69 D; P = .017), but refractive cylinder remained stable (from -2.83 to -2.61; P = .34). An improvement tendency of all anterior keratometric values was observed. A mean improvement of 120 µm in all thickness parameters was confirmed (P = .008), as well as an improvement in the spherical aberration (P = .018), coma (P = .23) and total higher order aberrations (P = .31). No significant differences among groups were detected. CONCLUSIONS: Decellularized human corneal stromal laminas transplantation seems safe and moderately effective for advanced keratoconus. Potential benefits of its recellularization with autologous adipose-derived adult stem cells remains unclear.

Cellular Therapy With Human Autologous Adipose-Derived Adult Stem Cells for Advanced Keratoconus.

PURPOSE: The aim of this phase 1 study was to preliminarily evaluate the safety and efficacy of autologous adipose-derived adult stem cell (ADASC) implantation within the corneal stroma of patients with advanced keratoconus. METHODS: Five consecutive patients were selected. Autologous ADASCs were obtained by elective liposuction. ADASCs (3 × 10) contained in 1 mL saline were injected into the corneal stroma through a femtosecond-assisted 9.5-mm diameter lamellar pocket under topical anesthesia. Patients were reviewed at 1 day, 1 week, 1, 3, and 6 months postoperatively. Visual function, manifest refraction, slit-lamp biomicroscopy, intraocular pressure, endothelial cell density, corneal topography, corneal optical coherence tomography, and corneal confocal biomicroscopy were recorded. RESULTS: No intraoperative or postoperative complications were recorded, with full corneal transparency recovery within 24 hours. Four patients completed the full follow-up. All patients improved their visual function (mean: 1 line of unaided and spectacle-corrected distance vision and 2 lines of rigid contact lens distance vision). Manifest refraction and topographic keratometry remained stable. Corneal optical coherence tomography showed a mean improvement of 16.5 µm in the central corneal thickness, and new collagen production was observed as patchy hyperreflective areas at the level of the stromal pocket. Confocal biomicroscopy confirmed the survival of the implanted stem cells at the surgical plane. Intraocular pressure and endothelial cell density remained stable. CONCLUSIONS: Cellular therapy of the human corneal stroma in vivo with autologous ADASCs appears to be safe. Stem cells survive in vivo with intrastromal new collagen production. Future studies with larger samples are required to confirm these preliminary results.