Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line

Inoue, Yu; Hasegawa, Seiji; Miyachi, Katsuma; Yamada, Takaaki; Nakata, Satoshi; Ipponjima, Sari; Hibi, Terumasa; Nemoto, Tomomi; Tanaka, Masahiko; Suzuki, Ryo; Hirashima, Naohide

doi:10.1111/exd.13672

Cited by 6 publications

(10 citation statements)

References 21 publications

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“…[ 30 ], were used as an epidermal stem/progenitor cell model. HDK1 cells have been reported to be epidermal stem cells with a potential to form three-dimensional (3D) reconstructed epidermis in culture and positive for Integrin beta-1 (ITGB1) and Integrin alpha-6 (ITGA6) that are both known as epidermal stem cell markers [ 31 , 32 ]. SF8428 line established by Ban S et al.…”

Section: Methodsmentioning

confidence: 99%

“…HDK1 cells were induced to differentiate and generate 3D epidermis as described in Inoue et al. [ 31 ]. To examine the effect of GREM2 on the differentiation of HDK1 cells, human recombinant GREM2 Protein (NOVUS Biologicals, CO, USA) was added to the epidermal differentiation medium at final concentrations of 12.5 and 25 ng/mL, and cells were cultured for seven days.…”

Section: Methodsmentioning

confidence: 99%

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Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

Kawagishi‐Hotta

Hasegawa

Inoue

et al. 2021

Regenerative Therapy

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Introduction The skin is comprised of various kinds of cells and has three layers, the epidermis, dermis and subcutaneous adipose tissue. Stem cells in each tissue duplicate themselves and differentiate to supply new cells that function in the tissue, and thereby maintain the tissue homeostasis. In contrast, senescent cells accumulate with age and secrete senescence-associated secretory phenotype (SASP) factors that impair surrounding cells and tissues, which lowers the capacity to maintain homeostasis in each tissue. Previously, we found Gremlin 2 (GREM2) as a novel SASP factor in the skin and reported that GREM2 suppressed the differentiation of adipose-derived stromal/stem cells. In the present study, we investigated the effects of GREM2 on stem cells in the epidermis and dermis. Methods To examine whether GREM2 expression and the differentiation levels in the epidermis and dermis are correlated, the expressions of GREM2, stem cell markers, an epidermal differentiation marker Keratin 10 (KRT10) and a dermal differentiation marker type 3 procollagen were examined in the skin samples (n = 14) randomly chosen from the elderly where GREM2 expression level is high and the individual differences of its expression are prominent. Next, to test whether GREM2 affects the differentiation of skin stem cells, cells from two established lines (an epidermal and a dermal stem/progenitor cell model) were cultured and induced to differentiate, and recombinant GREM2 protein was added. Results In the human skin, the expression levels of GREM2 varied among individuals both in the epidermis and dermis. The expression level of GREM2 was not correlated with the number of stem cells, but negatively correlated with those of both an epidermal and a dermal differentiation markers. The expression levels of epidermal differentiation markers were significantly suppressed by the addition of GREM2 in the three-dimensional (3D) epidermis generated with an epidermal stem/progenitor cell model. In addition, by differentiation induction, the expressions of dermal differentiation markers were induced in cells from a dermal stem/progenitor cell model, and the addition of GREM2 significantly suppressed the expressions of the dermal differentiation markers. Conclusions GREM2 expression level did not affect the numbers of stem cells in the epidermis and dermis but affects the differentiation and maturation levels of the tissues, and GREM2 suppressed the differentiation of stem/progenitor cells in vitro . These findings suggest that GREM2 may contribute to the age-related reduction in the capacity to maintain skin homeostasis by suppressing the differentiation of epidermal and dermal stem/progenitor cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin

Kawagishi‐Hotta

Hasegawa

Inoue

et al. 2021

Regenerative Therapy

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“…2.6 | Three-dimensional (3D) human keratinocyte culture HDK1 cells transfected with an expression vector of tdTomato 27 and untransfected HDK1 cells were mixed at a 1:5 or 1:100 ratio. These mixed cells were seeded on Falcon ® Cell Culture Inserts (polyethylene terephthalate; pore size, 0.4 μm) at a density of 2 × 10 5 cells/insert in 0.4 ml of expansion medium (CnT-07; CELLnTEC).…”

Section: Quantitative Real-time Pcr Analysis (Qrt-pcr)mentioning

confidence: 99%

“…To visualize the behaviour of epidermal cells, first we introduced a transgene to express tdTomato into HDK1 cells. 27 Cellular senescence was induced by UVB irradiation in the HDK1 cells that were fluorescently labelled with tdTomato (S-tdHDK1), and unirradiated normal HDK1 cells labelled with td-Tomato (N-tdHDK1) were used as a control. These S-tdHDK1 or N-tdHDK1 cells were mixed with HDK1 cells that were neither fluorescent-labelled nor irradiated with UVB at a ratio of 1:100, and they were used to develop a three-dimensional epidermis in vitro (Figure 2B).…”

Section: Senescent Cells Are Preferentially Removed From the Surrounding Tissue By Interaction With Adjacent Cells Via Notch Signallingmentioning

confidence: 99%

Senescent cell removal via JAG1‐NOTCH1 signalling in the epidermis

Yoshioka¹,

Yamada

Hasegawa

et al. 2021

Experimental Dermatology

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Cellular senescence is a phenomenon characterized by irreversible cell cycle arrest. The shortening of telomeres results in cellular senescence and the replicative limit of a cell, as reported by Hayflick. 1 In addition, other reasons such as failure in regulating tumor genes or tumor suppressor genes, DNA damage caused by ionizing radiation or ultraviolet ray (UV) and oxidative stress by reactive oxygen species (ROS) production are also known to cause cellular senescence. 2 Senescent cells increase the expression of cyclin-dependent kinase inhibitors p21 (CDKN1A) and p16 (CDKN2A) and a tumor suppressor p53, and such cells stop the cell cycle. It is also known that senescent cells secrete inflammatory cytokines such as IL-1β, IL-6, a chemokine IL-8 and matrix metalloproteinases (MMPs). 3 Senescent cells reinforce senescence in an autocrine manner and also induce senescence in surrounding cells or damage them by paracrine signalling. These features are called senescence-associated secretory phenotype (SASP). [4][5][6][7] Senescent cells have been reported to increase

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“…Original studies employing alternative methods have been frequently published in Experimental Dermatology in the last years . In line with this tradition, the present special issue also showcases a number of original contributions, which can be viewed as current examples for new developments in alternative methods. The advances in reproducing human skin features in vitro make it possible to recapitulate also human skin diseases, thus replacing the use of animals and/or reducing their number by integrating in vivo and in vitro data.…”

mentioning

confidence: 99%