Impact of culture media on primary human corneal endothelial cells derived from old donors.

Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally.

Validation of airway porcine epithelial cells as an alternative to human in vitro preclinical studies.

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up.

Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas.

GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells.

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the ß-catenin and TGFß pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.

Lessons learnt, and still to learn, in first in human stem cell trials.

Developing cellular therapies is not straightforward. This Perspective summarizes the experience of a group of academic stem cell investigators working in different clinical areas and aims to share insight into what we wished we knew before starting. These include (1) choosing the stem cell line and assessing the genome of both the starting and final product, (2) familiarity with GMP manufacturing, reagent validation, and supply chain management, (3) product delivery issues and the additional regulatory challenges, (4) the relationship between clinical trial design and preclinical studies, and (5) the market approval requirements, pathways, and partnerships needed.

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium.

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction.

The Growing Medical Need for Tracheal Replacement: Reconstructive Strategies Should Overcome Their Limits.

Breathing, being predominantly an automatic action, is often taken for granted. However, respiratory diseases affect millions of people globally, emerging as one of the major causes of disability and death overall. Among the respiratory dysfunctions, tracheal alterations have always represented a primary challenge for clinicians, biologists, and engineers. Indeed, in the case of wide structural alterations involving more than 50% of the tracheal length in adults or 30% in children, the available medical treatments are ineffective or inapplicable. So far, a plethora of reconstructive approaches have been proposed and clinically applied to face this growing, unmet medical need. Unfortunately, none of them has become a well-established and routinely applied clinical procedure to date. This review summarizes the main clinical reconstructive attempts and classifies them as non-tissue engineering and tissue engineering strategies. The analysis of the achievements and the main difficulties that still hinder this field, together with the evaluation of the forefront preclinical experiences in tracheal repair/replacement, is functional to promote a safer and more effective clinical translation in the near future.

SOX2 Is a Univocal Marker for Human Oral Mucosa Epithelium Useful in Post-COMET Patient Characterization.

Total bilateral Limbal Stem Cells Deficiency is a pathologic condition of the ocular surface due to loss or impairment of corneal stem cell function, altering homeostasis of the corneal epithelium. Cultivated Oral Mucosa Epithelial Transplantation (COMET) is the only autologous treatment for this pathology. During the follow-up, a proper characterization of the transplanted oral mucosa on the ocular surface supports understanding the regenerative process. The previously proposed markers for oral mucosa identification (e.g., keratins 3 and 13) are co-expressed by corneal and conjunctival epithelia. Here, we propose a new specific marker to distinguish human oral mucosa from the epithelia of the ocular surface. We compared the transcriptome of holoclones (stem cells) from the human oral mucosa, limbal and conjunctival cultures by microarray assay. High expression of SOX2 identified the oral mucosa vs. cornea and conjunctiva, while PAX6 was highly expressed in corneal and conjunctival epithelia. The transcripts were validated by qPCR, and immunological methods identified the related proteins. Finally, the proposed markers were used to analyze a 10-year follow-up aniridic patient treated by COMET. These findings will support the follow-up analysis of COMET treated patients and help to shed light on the mechanism of corneal repair and regeneration.

Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity.

The corneal endothelium is the inner corneal mono-layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub-confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT-PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFß in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.

Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.

Regenerative Medicine of Epithelia: Lessons From the Past and Future Goals.

This article explores examples of successful and unsuccessful regenerative medicine on human epithelia. To evaluate the applications of the first regenerated tissues, the analysis of the past successes and failures addresses some pending issues and lay the groundwork for developing new therapies. Research should still be encouraged to fill the gap between pathologies, clinical applications and what regenerative medicine can attain with current knowledge.

A fine-tuned ß-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis.

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo-G0/G1) and proliferating (in vitro-G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed ß-catenin and transforming growth factor (TGF-ß) pathways to be correlated with the identified proteins. Treatment of rCEnC with a ß-catenin activator and inhibitor showed that ß-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-ß were regulated through ß-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of ß-catenin and TGF-ß.

Surgery Versus ATMPs: An Example From Ophthalmology.

Advanced therapy medicinal products (ATMPs) are the new frontier of medicine. Advanced therapy medicinal products are set out to satisfy unmet medical needs and provide new innovative, cutting-edge therapies for serious or life-threatening diseases, thus providing new therapeutic options for people with few or no possibility of treatment. They are divided into four groups including gene therapy medicinal products, cell-based therapy medicinal products, tissue-engineered products, and combined ATMPs, which in Europe refer to products that incorporate one or more medical devices with any of the previously mentioned ATMPs as part of the advanced medicine product (AIFA, 2017; Ten Ham et al., 2018). Advanced therapy medicinal products can potentially have long-term benefits, thus bringing a long-lasting positive impact on patient health. Advanced therapy medicinal product therapies are often administered just once or twice, which gives patients the possibility to heal quickly compared to traditional therapies. They also provide a long-term saving opportunity, both in terms of costs of treatments and procedures that are no longer necessary and in terms of quality of life and productivity. The resolution of the patient's illness has a monetary impact on the patient, the patient's caretakers, and especially on the society (Alliance for Regenerative Medicine, 2019). The aim of this paper was to provide an overview on the use of ATMPs approved in Europe, with a focus on blindness and visual impairment and the related economic burden. In this case study, the effective cost of a blind patient in different European countries was compared after treatment with ATMPs or traditional therapies, focusing on visual impairment caused by corneal opacity. Our evaluation includes an overview of the global economic impact of the two types of therapies on the society. We estimated direct healthcare costs, direct non-healthcare costs, and labor productivity losses, to include costs on healthcare, services, patients, their families and for the society in general. We could conclude that the costs of the two therapeutic approaches are comparable.

Retinoic acid/calcite micro-carriers inserted in fibrin scaffolds modulate neuronal cell differentiation.

The controlled release of cell differentiating agents is crucial in many aspects of regenerative medicine. Here we propose the use of hybrid calcite single crystals as micro-carriers for the controlled and localized release of retinoic acid, which is entrapped within the crystalline lattice. The release of retinoic acid occurs only in the proximity of stem cells, upon dissolution of the calcite hybrid crystals that are dispersed in the fibrin scaffold. These hybrid crystals provide a sustained dosage of the entrapped agent. The environment provided by this composite scaffold enables differentiation towards neuronal cells that form a three-dimensional neuronal network.

Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies.

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.

Advances in stem cell research and therapeutic development.

Despite many reports of putative stem-cell-based treatments in genetic and degenerative disorders or severe injuries, the number of proven stem cell therapies has remained small. In this Review, we survey advances in stem cell research and describe the cell types that are currently being used in the clinic or are close to clinical trials. Finally, we analyse the scientific rationale, experimental approaches, caveats and results underpinning the clinical use of such stem cells.

Laminin 332-Dependent YAP Dysregulation Depletes Epidermal Stem Cells in Junctional Epidermolysis Bullosa.

Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and α6ß4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB.

Global Consensus on Definition, Classification, Diagnosis, and Staging of Limbal Stem Cell Deficiency.

PURPOSE: Despite extensive knowledge gained over the last 3 decades regarding limbal stem cell deficiency (LSCD), the disease is not clearly defined, and there is lack of agreement on the diagnostic criteria, staging, and classification system among treating physicians and research scientists working on this field. There is therefore an unmet need to obtain global consensus on the definition, classification, diagnosis, and staging of LSCD. METHODS: A Limbal Stem Cell Working Group was first established by The Cornea Society in 2012. The Working Group was divided into subcommittees. Four face-to-face meetings, frequent email discussions, and teleconferences were conducted since then to obtain agreement on a strategic plan and methodology from all participants after a comprehensive literature search, and final agreement was reached on the definition, classification, diagnosis, and staging of LSCD. A writing group was formed to draft the current manuscript, which has been extensively revised to reflect the consensus of the Working Group. RESULTS: A consensus was reached on the definition, classification, diagnosis, and staging of LSCD. The clinical presentation and diagnostic criteria of LSCD were clarified, and a staging system of LSCD based on clinical presentation was established. CONCLUSIONS: This global consensus provides a comprehensive framework for the definition, classification, diagnosis, and staging of LSCD. The newly established criteria will aid in the correct diagnosis and formulation of an appropriate treatment for different stages of LSCD, which will facilitate a better understanding of the condition and help with clinical management, research, and clinical trials in this area.