Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions

Zamudio, Aldo C.; Wang, Zheng; Chung, So‐Hyang; Wolosin, J. Mario

doi:10.1016/j.exer.2015.10.021

Cited by 8 publications

(3 citation statements)

References 37 publications

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“…Zamudio and collaborators have proposed the use of TGF-β inhibitors (SB431542 or A-83-01 [190]) as a means to promote stem cell proliferation [191]. Limbal stem cell deficiency (LSCD) is a devastating corneal pathology that arises from the depletion of limbal epithelial stem cells (LESCs) that reside in the limbus and are crucial for maintaining corneal epithelial turnover and transparency.…”

Section: Therapeutic Targets For Tgf-β Signaling Based Therapiesmentioning

confidence: 99%

TGF-β-Based Therapies for Treating Ocular Surface Disorders

Ogata,

Verma,

Coulson-Thomas

et al. 2024

Cells

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The cornea is continuously exposed to injuries, ranging from minor scratches to deep traumas. An effective healing mechanism is crucial for the cornea to restore its structure and function following major and minor insults. Transforming Growth Factor-Beta (TGF-β), a versatile signaling molecule that coordinates various cell responses, has a central role in corneal wound healing. Upon corneal injury, TGF-β is rapidly released into the extracellular environment, triggering cell migration and proliferation, the differentiation of keratocytes into myofibroblasts, and the initiation of the repair process. TGF-β-mediated processes are essential for wound closure; however, excessive levels of TGF-β can lead to fibrosis and scarring, causing impaired vision. Three primary isoforms of TGF-β exist—TGF-β1, TGF-β2, and TGF-β3. Although TGF-β isoforms share many structural and functional similarities, they present distinct roles in corneal regeneration, which adds an additional layer of complexity to understand the role of TGF-β in corneal wound healing. Further, aberrant TGF-β activity has been linked to various corneal pathologies, such as scarring and Peter’s Anomaly. Thus, understanding the molecular and cellular mechanisms by which TGF-β1-3 regulate corneal wound healing will enable the development of potential therapeutic interventions targeting the key molecule in this process. Herein, we summarize the multifaceted roles of TGF-β in corneal wound healing, dissecting its mechanisms of action and interactions with other molecules, and outline its role in corneal pathogenesis.

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Section: Therapeutic Targets For Tgf-β Signaling Based Therapiesmentioning

confidence: 99%

TGF-β-Based Therapies for Treating Ocular Surface Disorders

Ogata,

Verma,

Coulson-Thomas

et al. 2024

Cells

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“…prion disease), the development of tumorigenic effects, and immune reactions. 25 , 34 , 49 , 50 Furthermore, in order to protect public health when transplanting processed medical products into humans, these techniques must be performed under good laboratory practices, a requirement which has been passed into law in many European countries. 39 , 43 , 51 …”

Section: Introductionmentioning

confidence: 99%

Limbal Stem Cell Deficiency and Treatment with Stem Cell Transplantation

Selver¹,

Yağcı²,

Eğrilmez³

et al. 2017

tjo

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The cornea is the outermost tissue of the eye and it must be transparent for the maintenance of good visual function. The superficial epithelium of the cornea, which is renewed continuously by corneal stem cells, plays a critical role in the permanence of this transparency. These stem cells are localized at the cornea-conjunctival transition zone, referred to as the limbus. When this zone is affected/destroyed, limbal stem cell deficiency ensues. Loss of limbal stem cell function allows colonization of the corneal surface by conjunctival epithelium. Over 6 million people worldwide are affected by corneal blindness, and limbal stem cell deficiency is one of the main causes. Fortunately, it is becoming possible to recover vision by autologous transplantation of limbal cells obtained from the contralateral eye in unilateral cases. Due to the potential risks to the donor eye, only a small amount of tissue can be obtained, in which only 1-2% of the limbal epithelial cells are actually limbal stem cells. Vigorous attempts are being made to expand limbal stem cells in culture to preserve or even enrich the stem cell population. Ex vivo expanded limbal stem cell treatment in limbal stem cell deficiency was first reported in 1997. In the 20 years since, various protocols have been developed for the cultivation of limbal epithelial cells. It is still not clear which method promotes effective stem cell viability and this remains a subject of ongoing research. The most preferred technique for limbal cell culture is the explant culture model. In this approach, a small donor eye limbal biopsy is placed as an explant onto a biocompatible substrate (preferably human amniotic membrane) for expansion. The outgrowth (cultivated limbal epithelial cells) is then surgically transferred to the recipient eye.Due to changing regulations concerning cell-based therapy, the implementation of cultivated limbal epithelial transplantation in accordance with Good Laboratory Practice using xenobiotic-free systems is becoming widely accepted both in Turkey and worldwide.

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“…The studies in the ocular surface epithelia have been facilitated by the remarkably high levels of ABCG2 expressed by the cells of the limbal outgrowth [14, 15]. However, efflux rate measurements in cells expressing exclusively ABCG2 or ABCB1 have shown that JC1 can serve as substratum for either multidrug resistance (MDR) transporter [16–18].…”

Section: Introductionmentioning

confidence: 99%

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry

2017

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The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facilitated the surface marker-independent isolation of live stem cells. A drawback, though, is the known toxicity of Hoechst dyes. In this study we show that JC1, a bathochromic mitochondrial membrane potential-sensitive dye applied at proper concentration, can yield flow cytometry fluorescent emission bivariate plots containing a low JC1 accumulation (JC1low) cohort. Using a combination of multiple cell lines, ABC-transporter inhibitors and viral vector-driven insertion of the ABCG2 gene or ABCG2 and ABCB1 shRNAs we demonstrate that JC1low can be generated by either of the two aforementioned multidrug resistance transporters. Complete wash out of mitochondrial bound JC1 required more than 24 h. In spite of this tight binding, the dye did not affect either the mitochondrial membrane potentials or the proliferation rate. In contrast, contemporaneous with its nuclear accumulation, Hoechst 33342 or DVC, caused changes in the fluorescent emission of mitochondrial membrane potential sensitive dyes resembling the effects caused by the mitochondrial uncoupler FCCP. In a number of cell lines exposure to Hoechst resulted in marked slow-down of proliferation and abolition of ABCG2 transport activity during the subsequent 2 days but in K562 cells the exposure induced cell extended death. Overall, its lack of toxicity vis. a vis. the toxicity and genotoxicity of the DNA intercalating dyes makes JC1 an ideal tool for isolating live cells expressing high multidrug resistance transport activity.

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Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions

Cited by 8 publications

References 37 publications

TGF-β-Based Therapies for Treating Ocular Surface Disorders

TGF-β-Based Therapies for Treating Ocular Surface Disorders

Limbal Stem Cell Deficiency and Treatment with Stem Cell Transplantation

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry

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