Standardization of organoid culture for evaluation of melanogenesis induced by UVB, UVA and visible light

Olivatti, Thainá Oliveira Felicio; Alcântara, Giovana Piteri; Espósito, Ana Cláudia Cavalcante; Silva, Márcia Guimarães da; Miot, Hélio Amante

doi:10.1016/j.abd.2019.06.005

Cited by 5 publications

(3 citation statements)

References 34 publications

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“…Samples were sectioned longitudinally in two parts and stored in 10 mL of DMEM (Dulbecco’s Modified Eagle’s Medium, high glucose – D5796, Sigma Inc., United Kingdom) in a sterile transparent plastic bottle and kept at room temperature, according to the protocol set forth by Olivatti. 12 A part of the fragment was placed immediately in the dark, while the other was exposed to radiation, for a total UVB, UVA, and VL dose (effective in the tissue) of 166 mJ/cm 2 , 1.524 J/cm 2 , and 40 J/cm 2 (blue-violet light component), respectively. The fragments were irradiated with artificial sources of UVB (230 µW/cm 2 ; source: FS72T12/UVB/HO) for 12 min, UVA (1270 µW/cm 2 ; source: Phillips TL 100W/10R) for 6 min, and LED light (110 mW/cm 2 in the blue-violet band; source: GBRLUX 200W) for 20 min, at a standardized distance of 10 cm.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of ex vivo melanogenic response to UVB, UVA, and visible light in facial melasma and unaffected adjacent skin

Alcântara

Espósito

Olivatti

et al. 2020

Anais Brasileiros de Dermatologia

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Background The independent role of solar radiation in the differential melanogenesis between melasma and adjacent skin is unknown. Objectives To assess the melanogenic responses of skin with facial melasma and of the adjacent skin to UVB, UVA, and visible light, in an ex vivo model. Methods This was a quasi-experimental study involving 22 patients with melasma. Facial melasma and adjacent skin samples were collected and stored in DMEM medium, at room temperature. One fragment was placed under the protection from light, while another was exposed to UVB, UVA, and visible light (blue-violet component): 166 mJ/cm 2 , 1.524 J/cm 2 , and 40 J/cm 2 , respectively. Subsequently, all samples were kept for 72 hours in a dark environment and stained by Fontana-Masson to assess basal layer pigmentation, dendrites, and melanin granulation. Results Effective melanogenesis was observed in the basal layer in melasma and in the normal adjacent skin after all irradiations ( p < 0.01), with the following median increment: UVB (4.7% vs . 8.5%), UVA (9.5% vs . 9.9%), and visible light (6.8% vs . 11.7%), with no significant difference between anatomical sites. An increase in melanin granulation (coarser melanosomes) was observed only after irradiation with UVA and only in the skin with melasma ( p = 0.05). An increase in the melanocyte dendrite count induced by UVB radiation was observed in both anatomical sites ( p ≤ 0.05). Study limitations Use of an ex vivo model, with independent irradiation regimes for UVB, UVA, and visible light. Conclusions Melanogenesis induced by UVB, UVA, and visible light was observed both in melasma and in the adjacent skin. The morphological patterns suggest that different irradiations promote individualized responses on the skin with melasma.

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Section: Methodsmentioning

confidence: 99%

Evaluation of ex vivo melanogenic response to UVB, UVA, and visible light in facial melasma and unaffected adjacent skin

Alcântara

Espósito

Olivatti

et al. 2020

Anais Brasileiros de Dermatologia

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“…In addition to LMX1A, OTX2, and TH expression, at 30 dpt, the neuromelanin found in the grafted area was a direct indication of dopamine oxidation and, consequently, an indirect suggestion of dopamine synthesis by the grafted cells, which is an indispensable feature for dopaminergic neurons, as has also been seen in organoids directed towards a dopaminergic phenotype [42,43]. However, given that the transplant survived in only 16% (n = 18) of the rats at 30 dpt, there could be apoptotic signaling being triggered due to intrinsic characteristics of the cells (e.g., increased susceptibility to cell death as occurs in the dopaminergic phenotype), which could explain why at 7 and 15 dpt there was survival and differentiation as neural rosettes.…”

Section: Discussionmentioning

confidence: 84%

Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

Ramos-Acevedo

Morato-Torres

Bernal-Conde

et al. 2022

Preprint

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Background: Degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD) is responsible for motor and cognitive impairment. Replacing the dopaminergic cell population in the SNpc to restore normal dopamine levels is a potential therapeutic approach. However, improving neuronal integration still requires a reliable cell source for transplantation and a profound understanding of the effects of the local microenvironment on transplanted cells. We have previously shown that embryoid bodies (EBs)-derived cells from mouse embryonic stem cells overexpressing the dopaminergic transcription factor Lmx1a engrafted into SNpc develop tyrosine hydroxylase (TH)-positive phenotype. In the present work, we transplanted EBs-derived cells from genetically engineered human embryonic stem cells (hESCs), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO). We determined their potential to differentiate into TH-expressing neurons in the SNpc of an in vivo PD model. Methods: EBs-derived cells from genetically the engineered hESCs-LFO cell line were transplanted, and their neuronal differentiation potential was determined in the SNpc of an in vivo PD model with 6-hydroxy dopamine (6-OHDA). Three rat groups were designed as follows: Untreated (healthy rats), sham (rats administered with saline solution), and 6-OHDA (rats lesioned with 6-OHDA). A one-way ANOVA test was performed for statistical analysis. Results: Neural rosettes, a fundamental developmental hallmark of neuroepithelial tissue, were found at 7 and 15 days post-transplantation (dpt) in ~ 70% of the transplanted brains in all three conditions: Untreated, sham, and 6-OHDA. The majority of the neural rosettes corresponded to the lumen formation stage. In comparison, no graft survival was observed in EB transplants derived from unmodified hESCs. Interestingly, at 30 dpt, hESC-LFO engrafted cells showed neuronal morphology and positive immunolabeling for TH in all the brains exhibiting surviving transplants: 10% 6-OHDA rats, 0% sham, and 100% untreated rats. Conclusions: Overall, our results show that overexpression of LFO factors favors short-term survival while strongly initiating neural differentiation of hESC-derived cells in SNpc surviving grafts by forming neural rosettes and differentiating into TH-positive cells.

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“…Appropriate documentation of study design should be made to increase replicability and assist in troubleshooting.This technology does have several limitations due to the novelty of the method42 , mostly due to the lack of standardization in experimental design and execution of the protocol across laboratories. This lack of standardization has been acknowledged by other groups43 , and the Canine 3D Organoid Monolayer Protocols will lead to interlaboratory reproducibility and introduce standardization to this system. Standardized approaches to data analysis improve replicability and can strengthen results of preliminary drug testing using the canine organoids in the permeable supports system across different laboratories.…”

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confidence: 99%

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Gabriel

Zdyrski

Sahoo

et al. 2022

JoVE

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The permeable support system is typically used in conjunction with traditional twodimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol.Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol describes the tasks directly preceding an experiment, including in vitro validation of experimental results.

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Standardization of organoid culture for evaluation of melanogenesis induced by UVB, UVA and visible light

Cited by 5 publications

References 34 publications

Evaluation of ex vivo melanogenic response to UVB, UVA, and visible light in facial melasma and unaffected adjacent skin

Evaluation of ex vivo melanogenic response to UVB, UVA, and visible light in facial melasma and unaffected adjacent skin

Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

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