Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

Okumura, Naoki; Kusakabe, Ayaka; Hirano, Hiroatsu; Inoue, Ryota; Okazaki, Yugo; Nakano, Shinichiro; Kinoshita, Shigeru; Koizumi, Noriko

doi:10.1038/srep15005

Cited by 33 publications

(30 citation statements)

References 26 publications

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“…A total of four human donor corneas (from persons >40 years of age) were used for cultivation of HCECs by the protocol described previously. 19 Briefly, Descemet's membranes containing the HCECs were stripped from donor corneas, and the membranes were digested with 1 mg/mL collagenase A (Roche Applied Science, Penzberg, Germany) at 378C for 12 hours. The HCECs were seeded in 1 well of a 48-well plate coated with laminin E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) (0.5 lg/cm 2 ).…”

Section: Cell Culturementioning

confidence: 99%

Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing

Okumura

Okazaki

Inoue

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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117

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Section: Cell Culturementioning

confidence: 99%

Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing

Okumura

Okazaki

Inoue

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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117

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“…Recently, we have demonstrated that transplanted CECs of low cell density could regenerate corneal endothelium, but the cell density of the regenerated corneal endothelium was lower than that of the eye transplanted with high cell density CECs in a rabbit corneal endothelial dysfunction model. 18 These results suggested that obtaining high cell density CECs is essential for good prognosis after tissue engineering therapy.…”

Section: Discussionmentioning

confidence: 87%

“…However, the in vitro expansion of HCECs proved surprisingly difficult and we often had an insufficient number of cells with adequate cell quality for clinical use. 18 HCECs are difficult to maintain under culture conditions, and even if they survive, they show limited proliferative ability and undergo massive fibroblastic transformation with loss of functional phenotypes. Consequently, our research group and others have continuously strived to develop a successful culture method.…”

Section: Discussionmentioning

confidence: 99%

“…24 One remaining challenge is that HCECs tend to decrease their cell density, especially after several cell passages, leading to failure to obtain sufficient quantities of cells. 18 The CEC density is the most important indicator for corneal endothelial health in clinical settings, 25 so low cell density has become a concern with respect to transplanting cultured HCECs in humans. Recently, we have demonstrated that transplanted CECs of low cell density could regenerate corneal endothelium, but the cell density of the regenerated corneal endothelium was lower than that of the eye transplanted with high cell density CECs in a rabbit corneal endothelial dysfunction model.…”

Section: Discussionmentioning

confidence: 99%

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The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells

Hongo

Okumura

Nakahara

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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Citation: Hongo A, Okumura N, Nakahara M, Kay EP, Koizumi N. The effect of a p38 mitogen-activated protein kinase inhibitor on cellular senescence of cultivated human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2017;58:3325-3334. DOI:10.1167/iovs.16-21170 PURPOSE. We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs).METHODS. HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-b-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA.RESULTS. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 6 657 cells/mm 2 and 1752 6 628 cells/mm 2 , respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na þ /K þ -ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-b-gal staining, a low nuclear/ cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580.CONCLUSIONS. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

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“…32 To investigate the effect of culture protocols on morphologic differences in cultured HCEnCs, a series of primary cultures from different donors were investigated. Morphologically distinct subpopulations of highly uniform cells growing in colony-like structures, interspersed among largely nonproliferative and senescent primary cells, were detected in representative normal corneas from a 21-year-old man, 56-year-old man, and 70-year-old man (HCEnC-21M, HCEnC-56M, and HCEnC-70M, respectively) (Figure 1, AeC).…”

Section: Identification Of Highly Proliferative Cell Colonies From Nomentioning

confidence: 99%

Existence of Neural Crest–Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium

Katikireddy

Schmedt

Price

et al. 2016

The American Journal of Pathology

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Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crestederived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cellerelated genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75 NTR ), PAX3, SOX9, AP2B1 (AP-2b), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, beIII tubulin, and glial fibrillary acidic protein positive neurons and into p75 NTR -positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells on the posterior surface of the cornea and are essential for maintaining appropriate corneal hydration necessary for clear vision. HCEnCs sustain corneal clarity by serving as a barrier between the aqueous humor and the corneal stroma and by active ionic transport that regulates the swelling pressure of the cornea. HCEnCs are arrested in the postmitotic state and have limited proliferative capacity both in vivo and in vitro. The postmitotic arrest has been attributed to contact inhibition, transforming growth factor-b in aqueous fluid, and lack of paracrine stimulation by growth factors of cell-cycle promotion from the G 1 to the S phase.1 In a normal human life span, the endothelial cell density gradually declines.2 Therefore, age-and disease-related HCEnC loss is a major cause of corneal blindness requiring corneal transplantation to restore vision. Specifically, Fuchs endothelial corneal dystrophy (FECD) is the most common cause of endogenous endothelial cell dysfunction; it is associated with progressive cell apoptosis and concurrent extracellular matrix deposition in the form of guttae and is primarily treated by allogeneic endothelial keratoplasty.3,4 The pathogenic mechanism behind FECD is purportedly related to the interplay between genetic mutations and environmental factors. 5Several reports have proposed the existence of endothelial progenitor cells situated in the peripheral cornea, but evidence so far has not been conclusive. 6e8 The description of purported stem cells (SCs) has been based on ex vivo

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Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

Cited by 33 publications

References 26 publications

Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing

Effect of the Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) on Corneal Endothelial Wound Healing

The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells

Existence of Neural Crest–Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium

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