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)

Ett protokoll för att isolera, odla och identifiera humana limbala nischceller

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.

Författare i fokus: Standardisering av isolering och karakterisering av limbala nischceller (LNC) för att stödja omfattande LNC-forskning 

Isolering och identifiering av limbala nischceller

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)  

Identifiering av limbala nischceller (LNC)

För att identifiera de limbala nischcellerna eller LNC genom immunofluorescensfärgning, tillsätt 1 milliliter 0,25 % trypsin EDTA per brunn till LNC odlade i en 5 % matrigelbelagd 6-brunnsplatta. Smält cellerna genom att inkubera plattan i fem minuter vid 37 grader Celsius. Släck matsmältningen genom att tillsätta 1 milliliter knockoutserum som innehåller MESCM till cellsuspensionen.Överför cellsuspensionen till ett centrifugrör och centrifugera den 200 G i fem minuter innan supernatanten försiktigt aspireras. Återsuspendera pelleten i en milliliter MESCM. Använd sedan en cytofuge och deponera 80 mikroliter av cellsuspensionen lika mycket på fyra mikroskopglas enligt tillverkarens instruktioner.Fixera cellerna med 4% paraformaldehyd i cirka 10 minuter. Permeabilisera sedan cellerna på objektglasen med 0,2 % Triton X-100 i PBS i 15 minuter. Blockera cellerna med 2 % bovint serumalbumin i en timme innan du inkuberar dem med primära antikroppar på en shaker över natten vid 4 grader Celsius.Efter inkubationen avlägsnas obundna antikroppar genom att tvätta objektglasen med PBST tre gånger i fem minuter vardera. Därefter inkuberas provet med lämpliga sekundära antikroppar vid 37 grader Celsius i en timme innan eventuella obundna antikroppar tvättas som tidigare visats. Motfärga kärnorna med DAPI som har en koncentration på 5 mikrogram per milliliter.Efter att ha förseglat objektglasen, utför avbildning med hjälp av ett fluorescensmikroskop. Använd ett RNA-isoleringskit för att extrahera det totala RNA:t från LNC:erna enligt tillverkarens instruktioner. Använd sedan ett komplementärt DNA- eller CDNA-transkriptionskit med hög kapacitet och transkribera 1 till 2 mikrogram av RNA.Förbered en 20 mikroliters lösning som innehåller 2.5 mikroliter CDNA, 0.8 mikroliter motsvarande genetisk primer, 10 mikroliter av en universell PCR-masterblandning och 6.7 mikroliter dubbeldestillerat vatten. Utför sedan en kvantitativ polymeraskedjereaktion med lämpliga förhållanden. Slutligen används den jämförande CT-tekniken för att undersöka det relativa genuttrycket.Dubbel immunfärgning av de fjärde passage-LNC:erna avslöjade tydligt att dessa celler genomgående var panCK-negativa, VIM-positiva, CD90-positiva, CD105-positiva, SCF-positiva och PDGFR-positiva.

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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 ...