Name: Identification of Limbal Niche Cells (LNCs)
Uploaded: 2023-10-27T14:10:00
Description: Watch this Scientific Journal Video about Identification of Limbal Niche Cells LNCs at JoVE.com

identification of limbal niche cells (lncs)

Un protocollo per isolare, coltivare e identificare le cellule di nicchia limbare umana

The incidence of corneal epithelial stem cell deficiency (CESD), also called limbal stem cells deficiency (LSCD)1, and&#160;corneal epithelial regeneration (CES) are&#160;becoming more and more urgent because of corneal infection&#160;and injury. If not properly treated, CESD can lead to blindness that requires corneal transplantation. As a result, CES regeneration is&#160;becoming more significant. There is a group of supportive cells called limbal niche cells (LNCs) that provide essential support for CES function. Limbal stromal stem cells were first isolated by Polisetty et al.2 and identified by Xie et al.3&#160;as LNCs which are localized in the limbal epithelium subjacent and stroma of the limbus. LNCs are the key supporting stem cell of the corneal rim&#160;and, with the function of bone marrow-derived MSC (BMMSCs), and could&#160;be induced to develop into corneal epithelial cells and corneal stromal cells, etc.3,4,5,6,7. Previous studies showed that the stem-cell qualities of LNCs are more primitive than BMMSCs8, which is already widely used in the clinic. LNCs may even become the next viable option after MSC, especially for treating CESD. As important supporting cells for CES, LNCs are also stem cells derived from the &#34;niche&#34; structure of the limbus. LNCs may play a key role in the dedifferentiation of mature corneal epithelial cells (MCEC) to CES9. However, studies on LNCs are still relatively insufficient, and there is no consensus on the terminology, isolation, purification, identification, and characteristics of LNCs. Some researchers have named LNCs limbal biopsy-derived stromal stem cells10, limbal mesenchymal stem cells11, limbal fibroblast stem cells12, and limbal mesenchymal stromal cells13. As the growth characteristics of LNCs have not been described in detail, and because of their promising scientific and clinical applications, and may be one of the most important clinical tools in the future, it is necessary to summarize the isolation, purification, identification, and characteristics of LNCs.
According to a previous study14, LNCs are mainly present at the limbal epithelium subjacent and stroma of the limbus. This protocol includes treating limbus tissue using collagenase A, obtaining a cluster consisting of LEPC and LNCs, and digesting it into single cells with 0.25% trypsin-EDTA (TE). LNCs were then selectively cultured in a modified embryonic stem cell medium (MESCM) to be purified. The protocol reported in this paper is simple and has high efficiency in obtaining human LNCs in large quantities.
The&#160;detailed procedure of LNC isolation, culture, and identification was recorded in the video for scientists who are interested in LNC&#160;study, and it can be conveniently repeated when needed.

Autore in primo piano: Standardizzazione dell'isolamento e della caratterizzazione delle cellule di nicchia limbare (LNC) per supportare la ricerca diffusa sui LNC 

Isolamento e identificazione delle cellule di nicchia limbari

Our studies focus on the reconstruction of the limbal niche to treat limbal stem cell deficiency and to restore the patient's vision. We are trying to answer whether or not limbal niche cells are the key cells supporting the function of limbal stem cells, and if so, how it works. Recent studies reported that subconjunctival transplantation of mesenchymal stem cells from bone marrow prevented the limbal stem cell deficiency of alkali-burned eyes.One of the technologies currently used to advance our research is single cell sequencing, which can analyze the key signaling pathway activities at a single cell level for limbal niche cell characterizing. Although LNC have the potential to become an important clinical tool in the future, their growth characteristics have not been explored in much details. Considering their promising scientific and clinical applications, it is necessary to summarize the isolation, purification, identification, and characteristics of LNC.

Watch this Scientific Journal Video about Identification of Limbal Niche Cells LNCs at JoVE.com

Identification of Limbal Niche Cells (LNCs)  

Identificazione delle cellule di nicchia limbare (LNC)

Per identificare le cellule di nicchia limbare o LNC mediante colorazione a immunofluorescenza, aggiungere 1 millilitro di EDTA allo 0,25% di tripsina per pozzetto alle LNC coltivate in una piastra a 6 pozzetti rivestita di matrigel al 5%. Digerire le cellule incubando la piastra per cinque minuti a 37 gradi Celsius. Estinguere la digestione aggiungendo 1 millilitro di siero knockout contenente MESCM alla sospensione cellulare.Trasferire la sospensione cellulare in una provetta da centrifuga e centrifugarla a 200 G per cinque minuti prima di aspirare con cautela il surnatante. Risospendere il pellet in un millilitro di MESCM. Successivamente, utilizzando un citofugo, depositare 80 microlitri della sospensione cellulare in parti uguali su quattro vetrini da microscopio secondo le istruzioni del produttore.Fissare le cellule con paraformaldeide al 4% per circa 10 minuti. Quindi permeabilizzare le cellule sui vetrini utilizzando lo 0,2% di Triton X-100 in PBS per 15 minuti. Bloccare le cellule con albumina sierica bovina al 2% per un'ora prima di incubarle con gli anticorpi primari su uno shaker per una notte a 4 gradi Celsius.Dopo l'incubazione, rimuovere gli anticorpi non legati lavando i vetrini con PBST tre volte per cinque minuti ciascuna. Successivamente, incubare il campione con anticorpi secondari idonei a 37 gradi Celsius per un'ora prima di lavare eventuali anticorpi non legati, come dimostrato in precedenza. Contrastare i nuclei con DAPI avente una concentrazione di 5 microgrammi per millilitro.Dopo aver sigillato i vetrini, eseguire l'imaging utilizzando un microscopio a fluorescenza. Utilizzando un kit di isolamento dell'RNA, estrarre l'RNA totale dagli LNC secondo le istruzioni del produttore. Quindi, utilizzando un kit di trascrizione di DNA complementare o CDNA ad alta capacità, trascrivere inversamente da 1 a 2 microgrammi di RNA.Preparare una soluzione da 20 microlitri contenente 2,5 microlitri di CDNA, 0,8 microlitri di primer genetico corrispondente, 10 microlitri di una miscela master PCR universale e 6,7 microlitri di acqua bidistillata. Quindi eseguire una reazione a catena della polimerasi quantitativa utilizzando le condizioni appropriate. Infine, utilizzare la tecnica TC comparativa per esaminare l'espressione genica relativa.La doppia immunocolorazione degli LNC di quarto passaggio ha rivelato chiaramente che queste cellule erano costantemente panCK negative, VIM positive, CD90 positive, CD105 positive, SCF positive e PDGFR positive.

Research

JoVE Journal

Medicine

Identification of Limbal Niche Cells (LNCs)

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...