Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2)

Yamamoto, Naoki; Takeda, Shun; Hatsusaka, Natsuko; Hiramatsu, Noriko; Nagai, Noriaki; Deguchi, Saori; Nakazawa, Yosuke; Takata, Takumi; Kodera, Sachiko; Hirata, Akimasa; Kubo, Eri; Sekiya, Hiroshi

doi:10.3390/cells9122670

Cited by 11 publications

(8 citation statements)

References 64 publications

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“…The composition of the used LEC medium (with the STEP-4 culture condition being the same; Fig. 1 ), which induces H-iris cells to differentiate into LECs, was as follows ( 28 ): 10% (w/v) fetal bovine serum (cat. no.…”

Section: Methodsmentioning

confidence: 99%

Formation of three‑dimensional cell aggregates expressing lens‑specific proteins in various cultures of human iris‑derived tissue cells and iPS cells

et al. 2022

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Induced pluripotent stem (iPS) cells are widely used as a research tool in regenerative medicine and embryology. In studies related to lens regeneration in the eye, iPS cells have been reported to differentiate into lens epithelial cells (LECs); however, to the best of our knowledge, no study to date has described their formation of three-dimensional cell aggregates. Notably, in vivo studies in newts have revealed that iris cells in the eye can dedifferentiate into LECs and regenerate a new lens. Thus, as basic research on lens regeneration, the present study investigated the differentiation of human iris tissue-derived cells and human iris tissue-derived iPS cells into LECs and their formation of three-dimensional cell aggregates using a combination of two-dimensional culture, static suspension culture and rotational suspension culture. The results revealed that three-dimensional cell aggregates were formed and differentiated into LECs expressing αA-crystallin, a specific marker protein for LECs, suggesting that the cell-cell interaction facilitated by cell aggregation may have a critical role in enabling highly efficient differentiation of LECs. However, the present study was unable to achieve transparency in the cell aggregates; therefore, we aim to continue to investigate the degradation of organelles and other materials necessary to make the interior of the formed cell aggregates transparent. Furthermore, we aim to expand on our current work to study the regeneration of the lens and ciliary body as a whole in vitro, with the aim of being able to restore focusing function after cataract surgery.

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Section: Methodsmentioning

confidence: 99%

Formation of three‑dimensional cell aggregates expressing lens‑specific proteins in various cultures of human iris‑derived tissue cells and iPS cells

et al. 2022

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“…Cell lines. The HLE-immortalized cell lines SRA01/04 (RCB1591, Riken BRC, Tsukuba, Japan) and iHLEC-NY2 were donated by Professor Ibaraki (21) and Professor Yamamoto (22). Briefly, iHLEC-NY2 cells were derived from lens epithelial cells (LECs) collected during cataract surgery in adult humans.…”

Section: Methodsmentioning

confidence: 99%

“…LECs were cultured in Dulbecco's modified Eagle's medium (DMEM) high-glucose medium containing 10% fetal bovine serum (FBS). The immortalization gene was transferred to the pseudo-attachment P site of cultured LECs, and the cells were cloned to establish the immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2) ( 22 ). The use of iHLEC-NY2 cells was approved by the Fujita Medical University Recombinant DNA Experiment Safety Committee (approval no.…”

Section: Methodsmentioning

confidence: 99%

Function of mitochondrial cytochrome c oxidase is enhanced in human lens epithelial cells at high temperatures

et al. 2022

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Enhancement of density via human lens epithelium (HLE) cell proliferation is the underlying cause of nuclear cataracts. Moreover, our previous epidemiological study demonstrated that the risk of nuclear cataract development is significantly higher under elevated environmental temperatures compared with under lower temperatures. The present study investigated the relationship between temperature and cell proliferation in terms of mitochondrial function, which is a nuclear cataract-inducing risk factor, using two different HLE cell lines, SRA01/04 and immortalized human lens epithelial cells NY2 (iHLEC-NY2). Cell proliferation was significantly enhanced under the high-temperature condition (37.5˚C) in both cell lines. The cell growth levels of SRA01/04 and iHLEC-NY2 cells cultured at 37.5˚C were 1.20-and 1.16-fold those in the low-temperature cultures (35.0˚C), respectively. Moreover, the levels of cytochrome c oxidase mRNA (mitochondrial genome, cytochrome c oxidase-1-3) and its activity in SRA01/04 and iHLEC-NY2 cells cultured at 37.5˚C were higher compared with those in cells cultured at 35.0˚C. In addition, adenosine-5'-triphosphate (ATP) levels in SRA01/04 and iHLEC-NY2 cells were also significantly higher at 37.5˚C compared with those at 35.0˚C. By contrast, no significant differences in Na + /K + -ATPase or Ca 2+ -ATPase activities were observed between HLE cells cultured at 35.0 and 37.5˚C. These results suggested that expression of the mitochondrial genome was enhanced in high-temperature culture, resulting in a sufficient ATP content and cell proliferation for lens opacity. Therefore, elevated environmental temperatures may increase the risk of nuclear cataracts caused by HLE cell proliferation via mitochondrial activation.

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“…We prepared H-iris iPS cells by cell reprogramming using human iris tissue-derived p75NTR-positive stem/progenitor cells (H-iris stem/progenitor cells). The H-iris stem/progenitor cells were reprogrammed by a micro-electroporation method using an Epi5™ Episomal iPSC Reprogramming Kit (ThermoFisher Scientific) [ 31 , 32 ]. The reagent concentration of the kit was 2 μL/well (2 μg), and the concentration of the introduced acceleration reagent was also 2 μL/well (2 μg).…”

Section: Methodsmentioning

confidence: 99%

Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells

Yamamoto

Hiramatsu

Ohkuma

et al. 2021

Cells

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Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.

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Effect of a Lens Protein in Low-Temperature Culture of Novel Immortalized Human Lens Epithelial Cells (iHLEC-NY2)

Cited by 11 publications

References 64 publications

Formation of three‑dimensional cell aggregates expressing lens‑specific proteins in various cultures of human iris‑derived tissue cells and iPS cells

Formation of three‑dimensional cell aggregates expressing lens‑specific proteins in various cultures of human iris‑derived tissue cells and iPS cells

Function of mitochondrial cytochrome c oxidase is enhanced in human lens epithelial cells at high temperatures

Novel Technique for Retinal Nerve Cell Regeneration with Electrophysiological Functions Using Human Iris-Derived iPS Cells

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