Multilineage differentiation potential of cells isolated from the human amniotic membrane

[Abstract] The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM-derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion-derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well-defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem-cell markers SSEA-4 and STRO-1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-004

Intraneural IFG-1 in cryopreserved nerve isografts increase neural regeneration and functional recovery in the rat sciatic nerve

[Abstract] Background: Insulin-like growth factor 1 (IGF-1) was found to stimulate Schwann cell mitosis. Exogenous IGF-1 may improve nerve regeneration after cryopreservation. Objective: To evaulate the effect of intraneural administration of IGF-1 in cryopreserved nerve isografts. Methods: Eighteen millimeter grafts were used for bridging an 18-mm defect in the rat sciatic nerve. A total of 57 rats were randomly divided into three groups: (1) autograft (Group 1); (2) cryopreserved isograft (Group 2); (3) cryopreserved isograft with intraneural IGF-1 administration (Group 3). 12 weeks after surgery, functional recovery (Sciatic functional index [SFI], Swing speed [SS], nerve conduction velocity [NCV], amplitude of compound motor action potentials [CMAP], and gastrocnemius muscle index [GMI]) and nerve regeneration (myelin sheath area, total fiber counts, fiber density, and fiber width) were all evaluated. Results: The intraneural injection of IGF-1 significantly improved SFI and SS at weeks 10 and 12. There were no statistical differences between Groups 1 and 3 in any of the SFI or SS evaluations. CMAP and NCV in Group 1 were significantly higher than in Groups 2 and 3, and Group 3 had significantly higher CMAP and NCV compared to Group 2. No significant differences were found in fiber width. The number of nerve fibers, percentage of myelinated fibers, fiber density, and GMI was significantly higher in Group 1 compared to Group 2, but no significant differences were found between Groups 1 and 3. Conclusion: The results show that intraneural injection of IGF-1 in an 18 mm cryopreserved isograft improve axonal regeneration and functional recovery

Current Development of Alternative Treatments for Endothelial Decompensation: Cell-Based Therapy

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] Current treatment for corneal endothelial dysfunction consists in the replacement of corneal endothelium by keratoplasty. Owing to the scarcity of donor corneas and the increasing number of transplants, alternative treatments such as cell-based therapies are necessary. In this article, we highlight the biological aspects of the cornea and the corneal endothelium, as well as the context that surrounds the need for new alternatives to conventional keratoplasty. We then review some of those experimental treatments in more detail, focusing on the development of the in vitro and preclinical phases of two cell-based therapies: tissue-engineered endothelial keratoplasty (TE-EK) and cell injection. In the case of TE-EK graft construction, we analyse the current progress, considering all the requirements it must meet in order to be functional. Moreover, we discuss the inherent drawbacks of endothelial keratoplasties, which TE-EK grafts should overcome in order to make surgical intervention easier and to improve the outcomes of current endothelial keratoplasties. Finally, we analyse the development of preclinical trials and their limitations in terms of performing an optimal functional evaluation of cell-based therapy, and we conclude by discussing early clinical trials in humans.Xunta de Galicia; R2016/036Xunta de Galicia; ED431B 2020/55Xunta de Galicia; ED481B 2017/029Xunta de Galicia; ED481A-2019/206Xunta de Galicia; ED481A-2017/280This work was carried out thanks to funding from the Rede Galega de Terapia Celular 2016 (R2016/036) and Grupos con Potencial de Crecemento 2020 (ED431B 2020/55) both from Xunta de Galicia. This work was supported by one postdoctoral and two predoctoral fellowships from the Xunta de Galicia and the European Union (European Regional Development Fund) [grant numbers ED481B 2017/029, ED481A-2019/206, and ED481A-2017/280, respectively], as well as by two predoctoral fellowships for research stays from INDITEX-University of A Coruña-2019

Isolation and characterization of mesenchymal stem cells from human amniotic membrane

[Abstract] Introduction: The human amniotic membrane is a highly abundant and readily available tissue that may be useful for regenerative medicine and cell therapy. Aim: To compare two previously published protocols for the isolation of human amnion mesenchymal stromal cells (hAMSCs), including their phenotypic characterization and in vitro potential for differentiation toward osteogenic, adipogenic, and chondrogenic mesodermal lineages. Materials and Methods: Human placentas were obtained from selected caesarean-sectioned births. Two different protocols (Alviano et al.1 and Soncini et al.2) for the isolation of hAMSCs were performed. After monolayer expansion of adherent cells from both protocols, the cells were characterized by flow cytometry and for multi- potentiality, as assessed by their capability to differentiate toward adipocyte-, osteoblast-, and chondrocyte-like cells. Results: Both protocols yielded hAMSCs that showed plastic adherence, fibroblast-like growth, and well-defined human MSC markers. The cell yield and mesodermal differentiation capability of hAMSCs were higher in cells isolated using the Soncini protocol. Conclusions: Our data demonstrated the successful isolation of hAMSCs from full-term placentas using two published protocols. Differences between the two protocols in cell yield and in vitro differentiation potential are shown.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-004

Human amniotic membrane as an alternative source of stem cells for regenerative medicine

[Abstract] The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBNCB06-01-0040Instituto de Salud Carlos III; PI 08/2028Ministerio Ciencia e Innovacion; PLE2009-014

Cell viability assay in corneal endothelium

Resumen del póster publicado en el II Annual Meeting CINBIO abstracts book [Internet], p. 65[Abstract] Introduction: Endothelium is the inner layer of the cornea, which must be viable for transplanting. The limited availability of corneas makes necessary the developing of preservation techniques that allow a long storage without losing endothelial viability.Objectives: Optimization of a cell viability assay in preserved corneas.Methods: One half of an endothelium from a cornea that was storage in hypothermic conditions and an endothelium of a cryopreserved cornea were stained with LIVE/DEAD imaging kit and Hoechst. The other half of endothelium was the negative control. Corneal endothelia were imaged using a fluorescence microscope.Results: Four sort of cells were visualized on both endothelia: viable cells with high esterase activity, intermediate cells with low esterase activity, non-viable cells without esterase activity, and cells only stained by Hoechst. Conclusions: Triple stain is effectiveto detect different sort of cells in endothelium of preserved corneas, included viable cells, depending on their esterase enzymatic activity and on cell and nuclear membrane damage

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas

[Abstract] Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to −40 °C and 3 °C/min to −120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.This research was funded by Xunta de Galicia (grant no. ED431B 2020/55), by Xunta de Galicia and the European Union (European Regional Development Fund; grant nos. ED481A-2019/206 and ED481A-2017/280), and by INDITEX and the University of A Coruña (grant no. 2019)Xunta de Galicia; ED431B 2020/55Xunta de Galicia; ED481A-2019/206Xunta de Galicia; ED481A-2017/28

Potential use of the human amniotic membrane as a scaffold in human articular cartilage repair

[Abstract] The human amniotic membrane (HAM) is an abundant and readily obtained tissue that may be an important source of scaffold for transplanted chondrocytes in cartilage regeneration in vivo. To evaluate the potential use of cryopreserved HAMs as a support system for human chondrocytes in human articular cartilage repair. Chondrocytes were isolated from human articular cartilage, cultured and grown on the chorionic basement membrane side of HAMs. HAMs with chondrocytes were then used in 44 in vitro human osteoarthritis cartilage repair trials. Repair was evaluated at 4, 8 and 16 weeks by histological analysis. Chondrocytes cultured on the HAM revealed that cells grew on the chorionic basement membrane layer, but not on the epithelial side. Chondrocytes grown on the chorionic side of the HAM express type II collagen but not type I, indicating that after being in culture for 3–4 weeks they had not de-differentiated into fibroblasts. In vitro repair experiments showed formation on OA cartilage of new tissue expressing type II collagen. Integration of the new tissue with OA cartilage was excellent. The results indicate that cryopreserved HAMs can be used to support chondrocyte proliferation for transplantation therapy to repair OA cartilage.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-004