Corneal Endothelial Expansion Promoted by Human Bone Marrow Mesenchymal Stem Cell-Derived Conditioned Medium (original) (raw)
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Cells, 2021
This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory...
Establishment of functioning human corneal endothelial cell line with high growth potential
PloS one, 2012
Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+)- and K(+)-dependent ATPase (Na(+)/K(+)-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Rever...
Propagation of Human Corneal Endothelial Cells ? A Novel Dual Media Approach
Cell Transplantation, 2000
Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is the only option to reverse the blindness, but a global shortage of donor material remains an issue. There are immense clinical interests in the development of alternative treatment strategies to alleviate current reliance on donor materials. For such endeavors, ex vivo propagation of human corneal endothelial cells (hCECs) is required, but current methodology lacks consistency, with expanded hCECs losing cellular morphology to a mesenchymal-like transformation. In this study, we describe a novel dual media culture approach for the in vitro expansion of primary hCECs. Initial characterization included analysis of growth dynamics of hCECs grown in either proliferative (M4) or maintenance (M5) medium. Subsequent comparisons were performed on isolated hCECs cultured in M4 alone against cells expanded using the dual media approach. Further characterizations were performed using immunocytochemistry, quantitative real-time PCR, and gene expression microarray. At the third passage, results showed that hCECs propagated using the dual media approach were homogeneous in appearance, retained their unique polygonal cellular morphology, and expressed higher levels of corneal endothelium-associated markers in comparison to hCECs cultured in M4 alone, which were heterogeneous and fibroblastic in appearance. Finally, for hCECs cultured using the dual media approach, global gene expression and pathway analysis between confluent hCECs before and after 7-day exposure to M5 exhibited differential gene expression associated predominately with cell proliferation and wound healing. These findings showed that the propagation of primary hCECs using the novel dual media approach presented in this study is a consistent method to obtain bona fide hCECs. This, in turn, will elicit greater confidence in facilitating downstream development of alternative corneal endothelium replacement using tissue-engineered graft materials or cell injection therapy.
In Vitro Expansion of Corneal Endothelial Cells for Clinical Application: Current Update
Cornea, 2022
Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved .3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased a-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications. T he corneal endothelium (CE) forms a monolayer on the posterior surface of the cornea that actively pumps water from the corneal stroma into the aqueous humor 1,2. At birth the human CE contains ,5,000 cells/ mm 2 , but the cells are mitotically inactive and as a result cell density decreases throughout life 3,4. There is a rapid, non-linear decrease in cell density from the second trimester to ages 2-10, most likely due to the increase in the size of the cornea, followed by a slower, linear decrease in cell density due to cell aging and death 5,6. When CE damage, disease, or aging causes cell density to drop below ,500 cells/mm 2 , the CE can no longer pump enough water to compensate for diffusion into the cornea, resulting in stromal edema, corneal clouding and eventual vision loss 7. Transplantation of donor CE tissue, either as a full-thickness penetrating keratoplasty (PK) or as one of the several forms of endothelial keratoplasty, can restore CE function and corneal transparency 8-11. While successful, rejection and recurrence of CE cell loss remain common complications of these whole tissue/organ grafts 12-16. Further, these grafts require use of donated cadaveric tissue, which in many parts of the world is limited in availability or is entirely non-existent 14,16. Thus, there remains a critical need for new therapies to repair, regenerate or replace the CE in order to reverse corneal edema and restore normal vision. Currently, endothelial grafts constitute a 151 replacement of CE tissue with that of a cadaveric cornea. The number of such grafts produced by each donor eye could be increased significantly if CE cells were expanded in culture before grafting. Such an approach requires the ability to expand CE cells in a manner that maintains physiological CE function and a compatible carrier on which to transplant an engineered CE monolayer. Historically, cultured adult CE cells have been observed to undergo one or two population doublings in vitro, but rapidly become senescent or undergo endothelial to mesenchymal transition (EMT) to a fibroblastic phenotype 17-19. A number of studies have optimized culture media formulation 15 and supplemented with growth factors such as FGF2, EGF and NGF 1,20 to stimulate CEC growth. Additionally, the use of extracts from bovine corneal endothelial cells 21 ,or small molecules such as Rho kinase inhibitor Y27632 22-24 and ascorbic acid 2 phosphate 25,26 have been used to expand CE cells. Still other studies have investigated improving CE cell isolation 27-31 , using various extracellular matrix (ECM) proteins to improve CE cell attachment 27,32-34 , and immortalizing the CE cells with the SV40 T-antigen 30,31. All of these approaches have resulted in some measurable improvement in CE cell expansion in vitro, but none have achieved satisfactory results. Reproducibility, senescence, and EMT after expansion in vitro continue to pose significant barriers to generating enough CE cells for therapeutic applications.
Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas
PLoS ONE, 2011
Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donorto-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability. Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage. The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.
Concentration-dependent inhibition of angiogenesis by mesenchymal stem cells
…, 2009
Mesenchymal stem cells (MSCs), which potentially transdifferentiate into multiple cell types, are increasingly reported to be beneficial in models of organ system injury. However, the molecular mechanisms underlying interactions between MSCs and host cells, in particular endothelial cells (ECs), remain unclear. We show here in a matrigel angiogenesis assay that MSCs are capable of inhibiting capillary growth. After addition of MSCs to EC-derived capillaries in matrigel at EC: MSC ratio of 1:1, MSCs migrated toward the capillaries, intercalated between ECs, established Cx43-based intercellular gap junctional communication (GJC) with ECs, and increased production of reactive oxygen species (ROS). These events led to EC apoptosis and capillary degeneration. In an in vivo tumor model, direct MSC inoculation into subcutaneous melanomas induced apoptosis and abrogated tumor growth. Thus, our findings show for the first time that at high numbers, MSCs are potentially cytotoxic and that when injected locally in tumor tissue they might be effective antiangiogenesis agents suitable for cancer therapy. (Blood. 2009;113:4197-4205) (ROS). The combined effect of these responses was capillary destruction. Further, in an in vivo melanoma model, MSCs inhibited tumor growth by abrogating growth of the tumor vasculature. These results indicate a novel property of MSCs, namely as cytotoxic agents that inhibit the formation of neocapillary networks. Methods Reagents Cell culture. M199 medium, F-12 nutrient mixture medium, lipofectin, Opti-MEM, and antibiotics (50 U/mL penicillin G and 50 g/mL streptomycin) were obtained from Invitrogen (Carlsbad, CA). G418 were obtained from Calbiochem (San Diego, CA). Dulbecco modified Eagle medium (DMEM) was obtained from Mediatech (Herndon, VA). Fetal bovine serum (FBS) and bovine calf serum were obtained from Hyclone (Logan, UT). Fluorophores. Mito Tracker Deep Red (MTDR) (500 nM), calcein red-orange AM (13 M), Hoechst 33342 (5 g/mL), and dichlorodihydrofluorescein diacetate (DCFH-DA) (10 M) were obtained from Molecular Probes (Eugene, OR). Inhibitors. Glycerrhetinic acid (GA) (5 M), N-acetyl-L-cysteine (NAC) (10 mM), and polyethylene glycol polyethylene glycol-conjugated (PEG)-catalase (100 U/mL) were obtained from Sigma-Aldrich (St Louis, MO). Cx 43 peptides gap 26 (VCYDKSFPISHVR; 160 M), and gap 27 (SRPTEKTIFII; 190 M) were purchased from Alpha Diagnostic International (San Antonio, TX). Antibodies. Antibodies against CD11b/c (integrin ␣M and ␣X chains; macrophage common antigen), CD29 (integrin 1 chain), CD45 (leukocyte common antigen), CD54 (intracellular cell adhesion molecule-1; intercellular adhesion molecule 1 [ICAM-1]), CD59 (membrane attack complexinhibitory protein), and CD90 (Thy-1) were obtained from BD Biosciences (San Jose, CA). Antibodies against CD31 (platelet/endothelial cell adhesion molecule 1; PECAM1), CD34 (hematopoietic stem cells antigen), CD44 (homing cell adhesion molecule; H-CAM), p-Tyr, and vascular endothelial (VE)-cadherin polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Tie-2 monoclonal antibody was purchased from Santa Cruz. Anti-connexin 43 monoclonal antibody was
The effects of different culture media on human corneal endothelial cells
Investigative ophthalmology & visual science, 2014
To investigate the most appropriate media condition for the proliferation and functional maintenance of human corneal endothelial cells (HCECs). We cultured HCECs in traditional media (medium A or D) and in stem cell media (medium E or N). The morphology of the cells was evaluated by inverted microscopy. Collagen, type VIII, alpha 2 and sodium-potassium adenosine triphosphatase (Na(+)-K(+) ATPase) expression were analyzed as differentiation markers. Octamer-binding transcription factor 3/4, glial fibrillary acidic protein, nestin and β-catenin expression were evaluated as stem cell associated proteins. The cell proliferation rate was evaluated with a cell counting kit-8 assay. Wound healing assays were also performed. The transendothelial electrical potential difference (TEPD) value was used to estimate the endothelial cell permeability in vitro. The proliferation and morphology analyses demonstrated that there were significant differences between the media. The expression of differ...
GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells
bioRxiv (Cold Spring Harbor Laboratory), 2022
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 GSK3 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-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 CHIR99021 induced downregulation of EnMT markers (SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the -catenin and TGF pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT and brings 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.