Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy.

INTRODUCTION: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. METHODS: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 µm Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. RESULTS: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. CONCLUSIONS: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

Decellularized Graft for Repairing Severe Peripheral Nerve Injuries in Sheep.

BACKGROUND AND OBJECTIVES: Peripheral nerve injuries resulting in a nerve defect require surgical repair. The gold standard of autograft (AG) has several limitations, and therefore, new alternatives must be developed. The main objective of this study was to assess nerve regeneration through a long gap nerve injury (50 mm) in the peroneal nerve of sheep with a decellularized nerve allograft (DCA). METHODS: A 5-cm long nerve gap was made in the peroneal nerve of sheep and repaired using an AG or using a DCA. Functional tests were performed once a month and electrophysiology and echography evaluations at 6.5 and 9 months postsurgery. Nerve grafts were harvested at 9 months for immunohistochemical and morphological analyses. RESULTS: The decellularization protocol completely eliminated the cells while preserving the extracellular matrix of the nerve. No significant differences were observed in functional tests of locomotion and pain response. Reinnervation of the tibialis anterior muscles occurred in all animals, with some delay in the DCA group compared with the AG group. Histology showed a preserved fascicular structure in both AG and DCA; however, the number of axons distal to the nerve graft was higher in AG than in DCA. CONCLUSION: The decellularized graft assayed supported effective axonal regeneration when used to repair a 5-cm long gap in the sheep. As expected, a delay in functional recovery was observed compared with the AG because of the lack of Schwann cells.

A novel decellularized nerve graft for repairing peripheral nerve long gap injury in the rat.

Decellularized nerve allografts are an alternative to autograft for repairing severe nerve injuries, since they have higher availability and do not induce rejection. In this study, we have assessed the regenerative potential of a novel decellularization protocol for human and rat nerves for repairing nerve resections, compared to the gold standard autograft. A 15-mm gap in the sciatic nerve was repaired with decellularized rat allograft (DC-RA), decellularized human xenograft (DC-HX), or fresh autograft (AG). Electrophysiology tests were performed monthly to evaluate muscle reinnervation, whereas histological and immunohistochemical analyses of the grafts were evaluated at 4 months. A short-term study was also performed to compare the differences between the two decellularized grafts (DC-RA and DC-HX) in early phases of regeneration. The decellularization process eliminated cellularity while preserving the ECM and endoneurial tubules of both rat and human nerves. Higher amount of reinnervation was observed in the AG group compared to the DC-RA group, while only half of the animals of the DC-HX showed distal muscle reinnervation. The number of regenerating myelinated axons in the mid-graft was similar between AG and DC-RA and lower in DC-HX graft, but significantly lower in both DC grafts distally. At short term, fibroblasts repopulated the DC-RA graft, supporting regenerated axons, whereas an important fibrotic reaction was observed around DC-HX grafts. In conclusion, the decellularized allograft sustained regeneration through a long gap in the rat although at a slower rate compared to the ideal autograft, whereas regeneration was limited or even failed when using a decellularized xenograft.

"Off-the-Shelf" Nerve Matrix Preservation.

Background: Decellularized human nerves overcome the limitations of the current treatments for large peripheral nerve injuries. However, the use of decellularized nerves requires an "off-the-shelf" availability for useful and actual clinical application. In this study, we addressed the preservation of the native and decellularized human nerve matrix in an integrative approach for tissue scaffold production. Materials and Methods: For native nerve matrix preservation analysis, we used histological examination and immunofluorescence to examine the structure, biomechanical assays to evaluate the tensile strength and Young's modulus, and analyzed the extracellular matrix (ECM) composition using enzyme-linked immunosorbent assay (ELISA) and biochemical assays for laminin, collagen and sulfated glycosaminoglycans (sGAG). After decellularization, nuclear remnants and DNA content were evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining and the picogreen quantification assay, as well as immunofluorescence or ELISA for cell rests (S100 protein and myelin staining) evaluation. Decellularized cryopreserved scaffolds were assayed for biomechanics, ECM composition, and structural maintenance. Cytotoxicity assays were performed to evaluate the biocompatibility of the nerve matrix extracts after cryopreservation. Results: We compared different strategies for native nerve storage and found that preservation up to 7 days at 4°C in Roswell Park Memorial Institute medium maintained biomechanical properties, such as Young's modulus and tensile strength, along with the structure and ECM composition, regarding laminin, collagen, and sGAG. After a successful decellularization process, that eliminated cell remnants, nerve scaffolds were frozen in an "in house" formulated cryoprotectant, using an automatic controlled rate freezer. Nerve structure, ECM composition, and biomechanical properties were maintained before and after the freezing process in comparison with native nerves. The extracts of the nerve scaffolds after thawing were not cytotoxic and the freezing process sustained good viability in 3T3 cells (graphical abstract). Conclusion: Since our approach facilitates transport, storage, and provide a ready-to-use alternative, it could be used in a clinical application for the treatment of long-gap peripheral nerve injuries in regenerative medicine.

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration.

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD.

Nicotinamide Prevents Apolipoprotein B-Containing Lipoprotein Oxidation, Inflammation and Atherosclerosis in Apolipoprotein E-Deficient Mice.

The potential of nicotinamide (NAM) to prevent atherosclerosis has not yet been examined. This study investigated the effect of NAM supplementation on the development of atherosclerosis in a mouse model of the disease. The development of aortic atherosclerosis was significantly reduced (NAM low dose: 45%; NAM high dose: 55%) in NAM-treated, apolipoprotein (Apo)E-deficient mice challenged with a Western diet for 4 weeks. NAM administration significantly increased (1.8-fold) the plasma concentration of proatherogenic ApoB-containing lipoproteins in NAM high-dose (HD)-treated mice compared with untreated mice. However, isolated ApoB-containing lipoproteins from NAM HD mice were less prone to oxidation than those of untreated mice. This result was consistent with the decreased (1.5-fold) concentration of oxidized low-density lipoproteins in this group. Immunohistochemical staining of aortas from NAM-treated mice showed significantly increased levels of IL-10 (NAM low-dose (LD): 1.3-fold; NAM HD: 1.2-fold), concomitant with a significant decrease in the relative expression of TNFα (NAM LD: -44%; NAM HD: -57%). An improved anti-inflammatory pattern was reproduced in macrophages cultured in the presence of NAM. Thus, dietary NAM supplementation in ApoE-deficient mice prevented the development of atherosclerosis and improved protection against ApoB-containing lipoprotein oxidation and aortic inflammation.

Extrinsic modulation of integrin α6 and progenitor cell behavior in mesenchymal stem cells.

Mesenchymal stem cells (MSC) are heterogeneous cells of complex nature that show different potentials while different culture conditions can modify their functionalities through interactions with the microenviroment. Here, we found that bone marrow (BM) MSC from different donor sources and passages that expressed higher levels of α6 integrin subunit (ITGA6), showed higher clonogenicity, migration and differentiation potential. ITGA6 showed important roles improving these potentials and regulating proliferation through protein kinase B (AKT) pathway and cell cycle inhibitor proteins p53 and p21. Moreover, ITGA6 downregulation impaired migration. Cell confluence regulated ITGA6, increasing its expression in low density cultures and decreasing in high density cultures. Besides, ITGA6- cells expressed ITGA6 when seeded at low densities. We found higher ITGA6 expression on fibronectin substrates at lower confluency. Fibronectin increased proliferation, clonogenicity, activation of AKT, decreased cell cycle inhibitor proteins and augmented growth factors expression. Spheres-derived MSC showed higher ITGA6 expression and enhanced potentials for migration, clonogenicity and proliferation. In conclusion, though there is an intrinsic regulation of ITGA6 expression, associated to the progenitor potential of BM-MSC, this expression is regulated by culture conditions and is translated in changes in cell behavior and proliferation. This knowledge could be used to enhance the potential of BM-MSC for clinical application.

In vitro potential of human mesenchymal stem cells for corneal epithelial regeneration.

Aim: To determine the potential of mesenchymal stem cells (MSC) for corneal epithelial regeneration in vitro. Materials & methods: Bone marrow MSC (BM-MSC) and adipose tissue MSC were analyzed for corneal epithelial and mesenchymal markers, using limbal stem cells and corneal cells as controls. MSC with better potential were cultured with specific mediums for epithelial induction. Transepithelial electric resistance and wound healing assay with human corneal epithelial cells were performed. Results: BM-MSC showed better potential, increased corneal markers, and higher transepithelial electric resistance values when induced with limbal epithelial culture medium. Induced BM-MSC promoted better wound healing of human corneal epithelial cells by paracrine secretion. Conclusion: BM-MSC has potential for corneal epithelial induction in a protocol compatible with human application.

First-in-human PeriCord cardiac bioimplant: Scalability and GMP manufacturing of an allogeneic engineered tissue graft.

BACKGROUND: Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. METHODS: The PeriCord is a clinical-size (12-16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth. FINDINGS: PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area. INTERPRETATION: This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation. FUNDING: La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, "La Caixa" Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund.

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy.

BACKGROUND: Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives. METHODS: We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA. RESULTS: XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells. CONCLUSIONS: The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.

Limbal Stem Cells from Aged Donors Are a Suitable Source for Clinical Application.

Limbal stem cells (LSC) are the progenitor cells that maintain the transparency of the cornea. Limbal stem cell deficiency (LSCD) leads to corneal opacity, inflammation, scarring, and blindness. A clinical approach to treat this condition consists in LSC transplantation (LSCT) after ex vivo expansion of LSC. In unilateral LSCD, an autologous transplant is possible, but cases of bilateral LSCD require allogenic LSCT. Cadaveric donors represent the most important source of LSC allografts for treatment of bilateral LSCD when living relative donors are not available. To evaluate the suitability of aged cadaveric donors for LSCT, we compared three pools of LSC from donors of different ages (<60 years, 60-75 years, and >75 years). We evaluated graft quality in terms of percent of p63-positive (p63+) cells by immunofluorescence, colony forming efficiency, and mRNA and protein expression of p63, PAX6, Wnt7a, E-cadherin, and cytokeratin (CK) 12, CK3, and CK19. The results showed that LSC cultures from aged donors can express ≥3% of p63+ cells-considered as the minimum value for predicting favorable clinical outcomes after LSCT-suggesting that these cells could be a suitable source of LSC for transplantation. Our results also indicate the need to evaluate LSC graft quality criteria for each donor.

Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface.

The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement-cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET)-present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD.

Progenitor cells for ocular surface regenerative therapy.

The integrity and normal function of the corneal epithelium are essential for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio-replacement, such as cultured limbal epithelial transplantation and cultured oral mucosal epithelial transplantation, present very encouraging clinical results for treating limbal stem cell deficiencies. Another emerging therapeutic strategy consists of obtaining and implementing human progenitor cells of different origins using tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal stromal cells, represents a significant breakthrough in the treatment of certain eye diseases and also offers a more rational, less invasive and more physiological approach to ocular surface regeneration.