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 protocolo para aislar, cultivar e identificar células de nicho limbal humano

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.

Autor destacado: Estandarización del aislamiento y la caracterización de células de nicho limbal (LNC) para respaldar la investigación generalizada de LNC 

Aislamiento e identificación de células de nicho limbal

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)  

Identificación de células de nicho limbal (LNC)

Para identificar las células del nicho limbal o LNC mediante tinción de inmunofluorescencia, agregue 1 mililitro de EDTA de tripsina al 0,25 % por pocillo a los LNC cultivados en una placa de 6 pocillos recubierta de matrigel al 5 %. Digiere las células incubando la placa durante cinco minutos a 37 grados centígrados. Apague la digestión agregando 1 mililitro de suero knockout que contenga MESCM a la suspensión celular.Transfiera la suspensión celular a un tubo de centrífuga y centrifugue 200G durante cinco minutos antes de aspirar cuidadosamente el sobrenadante. Vuelva a suspender el pellet en un mililitro de MESCM. A continuación, utilizando un citofugio, deposite 80 microlitros de la suspensión celular en partes iguales en cuatro portaobjetos de microscopio según las instrucciones del fabricante.Fije las células con paraformaldehído al 4% durante aproximadamente 10 minutos. A continuación, permeabilice las células de los portaobjetos utilizando Triton X-100 al 0,2% en PBS durante 15 minutos. Bloquee las células con albúmina sérica bovina al 2% durante una hora antes de incubarlas con anticuerpos primarios en un agitador durante la noche a 4 grados centígrados.Después de la incubación, elimine los anticuerpos no unidos lavando los portaobjetos con PBST tres veces durante cinco minutos cada uno. A continuación, incubar la muestra con anticuerpos secundarios adecuados a 37 grados centígrados durante una hora antes de lavar los anticuerpos no unidos, como se ha demostrado anteriormente. Contratiñir los núcleos con DAPI que tenga una concentración de 5 microgramos por mililitro.Después de sellar los portaobjetos, realice la obtención de imágenes con un microscopio de fluorescencia. Usando un kit de aislamiento de ARN, extraiga el ARN total de los LNC según las instrucciones del fabricante. Luego, usando un kit de transcripción de ADN complementario o CDNA de alta capacidad, transcriba inversamente de 1 a 2 microgramos del ARN.Prepare una solución de 20 microlitros que contenga 2,5 microlitros del CDNA, 0,8 microlitros del cebador genético correspondiente, 10 microlitros de una mezcla maestra de PCR universal y 6,7 microlitros de agua bidestilada. A continuación, realice una reacción en cadena de la polimerasa cuantitativa utilizando las condiciones adecuadas. Por último, emplee la técnica de TC comparativa para examinar la expresión génica relativa.La doble tinción inmunológica de los LNC del cuarto paso reveló claramente que estas células eran consistentemente negativas para panCK, VIM positivas, CD90 positivas, CD105 positivas, SCF positivas y PDGFR positivas.

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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 ...