Preparation of Primary Cultures of Mouse Epidermal Keratinocytes and the Measurement of Phospholipase D Activity

ATP is known to stimulate surfactant phospholipid secretion in type II cells, and there is evidence that this effect is mediated by a P2 purinoceptor. At least five subtypes of the P2 receptor have been reported, but it is not clear which one exists on the type II cell. To determine whether it is the P2u subtype, at which UTP is equipotent with ATP, we have compared the effects of ATP and UTP on phosphatidylcholine secretion and second messenger formation in primary cultures of rat type II cells. ATP and UTP were equally potent in stimulating phosphatidylcholine secretion and phospholipase D activation. The potency order, UTP = ATP > ADP > 2-methylthio-ATP, was the same as that reported for the P2u receptor. UTP stimulated diacylglycerol and phosphatidic acid formation to the same extent as ATP. ATP also increased choline formation. Formation of diacylglycerol was biphasic, and the first peak in response to ATP was previously shown to be associated with inositol trisphosphate formation. Northern analysis showed that the P2u receptor gene was expressed to a greater extent in type II cells than in whole lung. These data suggest that ATP and UTP act via a P2u receptor that is coupled to phosphoinositide-specific phospholipase C with subsequent activation of phospholipase D acting on phosphatidylcholine. ATP has also been reported to act at an additional type II cell receptor coupled to adenylate cyclase. In contrast, UTP did not promote adenosine 3',5'-cyclic monophosphate formation and therefore does not act at that receptor.

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Gene Expression Profiling of Mouse Epidermal Keratinocytes

Methods in Molecular Biology - Epidermal Cells ◽

10.1007/978-1-60761-380-0_13 ◽

2009 ◽

pp. 171-181 ◽

Cited By ~ 1

Author(s):

Ramón García-Escudero ◽

Jesús M. Paramio

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Epidermal Keratinocytes ◽

Mouse Epidermal Keratinocytes

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An in vitro feasibility investigation considering primary human melanocytes for spray- grafting of freshly isolated autologous skin cells for burn treatment and a clinical case report

Surgical Case Reports ◽

10.31487/j.scr.2019.03.09 ◽

2019 ◽

pp. 1-8

Author(s):

Jörg C. Gerlach ◽

C. Johnen ◽

B. Hartmann ◽

J. Plettig ◽

K. Bräutigam ◽

...

Keyword(s):

Cell Morphology ◽

Skin Color ◽

In Vitro Model ◽

Primary Cultures ◽

Cell Suspensions ◽

Epidermal Keratinocytes ◽

Cell Counts ◽

Skin Coloration ◽

Vitro Model

A skin cell-spray grafting technique that enables the on-site application of freshly isolated autologous single cell suspensions was already applied in many cases on caucasian patients with low skin coloration. Our project hypothesis is that these suspensions contain keratinocytes and vital melanocytes, that are of particular interest for the treatment of patients of darker skin color. To test this, we applied an in vitro model, wherein the feasibility of i) isolating and ii) spraying of freshly isolated autologous melanocyte-keratinocyte cell suspensions was investigated. Primary human epidermal keratinocytes (HEKs) and melanocytes (MCs) were isolated from skin biopsies (n=8). Biochemical parameter, cell counts, cell morphology, growth behavior and immunofluorescence results were compared in two groups using MC cultures and co-cultures of MCs with HEKs. Case information on using the method clinically with one patient is included. The sprayed mixed cell suspensions proliferated in all groups without measurable loss of viability, and MCs exhibited a regular cell morphology in monoculture up to passage 4°. The sprayed MCs and HEKs demonstrated in vitro glucose and lactate metabolism that was comparable to the pipetted controls. In co-culture, well distributed CK14+ HEKs and NKI/beteb+ MCs could be demonstrated, which interacted in the in vitro model. The ratio of HEKs : MCs in our primary cultures were microscopically counted (n=8 each) as mean +/- SD 1,211,000 (+/- 574,343) HEK : 99,625 (+/- 59,025) MC; i.e., a ratio of approx. 12 : 1. Using the isolation method clinically for a patient with dark skin coloration after suffering severe second-degree burns shows a satisfying re-pigmentation of the resulting wound post healing. Freshly isolated spray-on melanocyte/keratinocyte suspensions provide for a considerable amount of viable HEKs and MCs. Using MCs in spray-grafting suspensions could represent a promising approach for treating severe partial-thickness burns and innovative therapy developments that also aim to address cosmetic aspects.

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Ionizing radiation, but not ultraviolet radiation, induces mitotic catastrophe in mouse epidermal keratinocytes with aberrant cell cycle checkpoints

Experimental Dermatology ◽

10.1111/exd.13665 ◽

2018 ◽

Vol 27 (7) ◽

pp. 791-794 ◽

Cited By ~ 2

Author(s):

Ming Wang ◽

QingXiang Gao ◽

Xu Teng ◽

MeiPing Pan ◽

TianMiao Lin ◽

...

Keyword(s):

Cell Cycle ◽

Ionizing Radiation ◽

Ultraviolet Radiation ◽

Cell Cycle Checkpoints ◽

Mitotic Catastrophe ◽

Epidermal Keratinocytes ◽

Aberrant Cell ◽

Mouse Epidermal Keratinocytes

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Members of the src and ras oncogene families supplant the epidermal growth factor requirement of BALB/MK-2 keratinocytes and induce distinct alterations in their terminal differentiation program

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3386-3396.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3386-3396

Author(s):

B Weissman ◽

S A Aaronson

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Terminal Differentiation ◽

Epidermal Keratinocytes ◽

Ras Oncogene ◽

Rapid Loss ◽

Naturally Occurring ◽

Epidermal Growth ◽

Transforming Genes ◽

Mouse Epidermal Keratinocytes

BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies.

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