Name: Isolation and Identification of Limbal Niche Cells (Video) | JoVE Journal
Uploaded: 2023-10-27T14:10:00.000+00:00
Duration: 10 min 11 s
Description: Learn isolation and identification of human limbal niche cells for treating limbal stem cell deficiency and restoring vision. This protocol supports regenerative medicine research using single cell sequencing.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Thanks to Wei Wang, Lingjuan Xu, and Rong Liu for the guidance on this work, Yongyao Tan, Bihui Jin, Chunxiu You, and Li Guigang for providing some of the material, Guanyu Su for writing the manuscript, Xiao Zhou, Yihong Xiong, and Huatao Xie for correcting the manuscript, and Guigang Li for his full guidance. This study was supported by the National Natural Science Foundation of China (No. 82070936, 81470606, 81570819), Hubei Province health and family planning scientific research project (No. WJ2017M073), Top Ten Translational Medical Research Projects from Tongji Hospital (No.2016ZHYX20), Training Project of Young medical Pioneers in Wuhan City (No.2015whzqnyxggrc10), Global Talents Recruitment Program (G2022154028L), National Health Commission of Hubei Province project In 2022(WJ2021ZH0005), and Subject Construction Foundation of Finance Department of Hubei In 2022(42000022815T000000102)

Limbus tissue from donors aged between 50 and 60 years was obtained from the Red Cross Eye Bank, Tongji Hospital (Wuhan, China). The protocol was approved by the Tongji Ethics Committee and was conducted in accordance with the Declaration of Helsinki.
1. Isolation
<ol>
	<li>Obtain limbus tissue from intermediate-term corneal storage medium and operate under aseptic conditions on an ultra-clean workbench.</li>
	<li>Scrape and remove the iris and endothelium around the cornea using a sterile surgical round blade.</li>
	<li>Cut limbus tissue into twelve equally sized pieces with a surgical blade with 1 mm left on both sides of the cornea&#160;and sclera.</li>
	<li>Transfer limbus tissues to a 3.5 cm culture plate, add 1 mL of collagenase A (2 mg/mL, in DF-12 medium), to immerse all of the tissues for digestion.</li>
	<li>Put the plate into the 37 &#176;C cell incubator, digest the limbal tissues for 18 h. 
	NOTE: Follow-up procedures are usually performed on the second day.</li>
	<li>Prepare a 5% basement membrane matrix (Matrigel) coated 6-well culture plate.
	<ol>
		<li>Coat 5% basement membrane matrix (dissolved in MESCM [Table 1]) at the bottom of the well.</li>
		<li>Prepare 200 &#956;L and 1 mL tips, one&#160;15-mL centrifugal tube, and one 6-well plate in a sterilized bag.</li>
		<li>Put the sterilized bag (including tips and centrifugal tube) and a new 6-well plate at -20 &#176;C or -80 &#176;C environment for 20 min. Thaw the basement membrane matrix in the&#160;refrigerator at 4 &#176;C.</li>
		<li>Take out the pre-cooled tips, centrifugal tube, and 6-well plate from the -20 &#176;C or -80 &#176;C environment, and then perform the following operations on the ultra-clean workbench.</li>
		<li>Transfer 50 &#181;L 5% basement membrane matrix to 1 mL MESCM using a 200 &#956;L tip, and mix them gently and evenly.</li>
		<li>Transfer 5% basement membrane matrix (dissolved in MESCM) to one well of a 6-well culture plate using the pre-cooled 1 mL tip.</li>
		<li>Shake the 6-well plate gently and horizontally, put the 6-well culture plate in the 37 &#176;C cell incubator for approximately 1 h, and then perform the next step.</li>
	</ol>
	</li>
	<li>After 18-h digestion, take out collagenase A-digested limbal tissue,&#160;only some visible small clusters and undigested scleral tissues will remain. 
	NOTE: High-magnification microscopy reveals a cluster composed of many densely packed cells (including LNCs).</li>
	<li>Use a 200 &#181;L pipette tip to detach the clusters from undigested scleral tissues under the stereomicroscope.</li>
	<li>Transfer the clusters to the 3.5-cm culture plate and immerse them in 0.25% TE, and then place the plate in the cell incubator for 15 min.</li>
	<li>Add 1 mL MESCM with 10% knockout serum to stop cell digestion and gently pipette up and down to resuspend the clusters into individual cells using a 1-mL pipette tip.</li>
	<li>Transfer the cell suspension to a 15-mL centrifugal tube and centrifuge at 200 x g for 5 min.</li>
	<li>Remove the supernatant carefully without disturbing the cell precipitation, and then add 1 mL MESCM to resuspend the cells.</li>
	<li>Pipette up and down 2-3 times until the bottom cell precipitation is evenly suspended in the MESCM using 1 mL pipette tip, and take 20 &#181;L cell suspension for cell counting.</li>
	<li>Transfer the cell suspension to the 5% basement membrane matrix-coated 6-well plate with a 1-mL pipette tip and increase the volume of MESCM to 2.5 mL.</li>
	<li>Gently shake the 6-well plate horizontally to evenly distribute the cells.</li>
	<li>Transfer the cells to the culture incubator after being imaged and recorded. Observe and photograph the cells daily, and change the medium every 3-4 days.</li>
</ol>
2. Identification of LNC
<ol>
	<li>Immunofluorescence staining
	<ol>
		<li>Digest the LNCs from the bottom of the plate using 1 mL 0.25% TE for 5 min in the&#160;37&#176;C cell incubator.</li>
		<li>Terminate the digestion by adding 1 mL MESCM (containing knockout serum), centrifuge the cell suspension at 200 x g for 5 min, and aspirate the supernatant carefully.</li>
		<li>Add 1 mL MESCM to resuspend the cells in suspension. Prepare 80 &#181;L cell suspension (4 &#215; 103 cells per slide) for 4 slides (20 &#181;L/slide) and use centrifuge system&#160;to deposit cells onto microscope slides as per the manufacturer&#39;s instructions.</li>
		<li>Fix cells (adhered to slides in step 3) using 4% paraformaldehyde for about 10 min, and then permeabilize cells on the slides with 0.2% Triton X-100 in PBS for 15 min.</li>
		<li>Block the cells for 1 h with 2% bovine serum albumin (BSA) before incubating them with primary antibodies (Pan-CK [1:1000], Vim [1:100], CD90 [1:100], CD105 [1:200], SCF [1:100], PDGFR&#946; [1:100]) on a shaker overnight at 4 &#176;C. After the incubation, remove unbound primary antibodies by washing (5 min/wash, 3x) the slides with PBST (PBS + 0.1% Tween 20).</li>
		<li>Incubate the corresponding secondary antibodies (donkey anti-mouse IgG secondary antibody TRITC [1:1000], donkey anti-rabbit IgG FITC [1:500], donkey anti-mouse IgG 488 [1:300], donkey anti-rabbit IgG 594 [1:400]) for 1 h with suitable IgG nonspecific IgG antibodies. Remove the unbound secondary antibody using PBST (5 min/wash, 3x).</li>
		<li>Counterstain the nuclei with 5 &#181;g/mL DAPI (4&#39;,6-Diamidino-2-Phenylindole ) and seal the slides.</li>
		<li>Perform fluorescence imaging using a fluorescence microscope (Magnification: 400x; Exposure time: 50 ms).</li>
	</ol>
	</li>
	<li>Quantitative real-time polymerase chain reaction (qPCR)
	<ol>
		<li>Take RNA Isolation Kit to extract the total RNA as per the manufacturer&#39;s instructions.</li>
		<li>Use a high-capacity cDNA Transcription Kit to reverse transcribe 1-2 &#181;g of total RNA to cDNA.</li>
		<li>Perform the reverse transcription (42 &#176;C, 2 min; 50 &#176;C , 85 min) and qPCR ( 95 &#176;C, 5 min; 95 &#176;C, 10 s, 40 cycles) in a 20-&#956;L solution containing cDNA (100 ng), TaqMan Gene Expression Assay Mix (1 &#956;L), universal PCR Master Mix (10 &#956;L) and ddH2O (to 20 &#181;L).</li>
		<li>Use the comparative CT technique (&#916;&#916;CT)4,9 to examine the relative gene expression.</li>
	</ol>
	</li>
</ol>
3. Characterization of LNC
<ol>
	<li>Cell counting (hemocytometer)
	<ol>
		<li>Digest the LNCs from the bottom of the plate using 0.25% TE&#160;for 5 min in the&#160;37 &#176;C cell incubator.</li>
		<li>Terminate the digestion by adding 1 mL MESCM (containing knockout serum), centrifuge the cell suspension at 200 x g for 5 min, and aspirate the supernatant carefully.</li>
		<li>Add 1 mL MESCM to resuspend the cells, then take 10 &#956;L cell suspension in a 0.5 mL centrifuge tube. Add an equal volume of 0.4% Trypan blue staining solution.</li>
		<li>Place 10 &#956;L stained cell suspension on the counting area of the hemocytometer and cover it with a coverslip for counting. 
		NOTE: The number of cells in the five middle square areas is defined as &#34;A&#34;; the cell number in 1 mL is calculated as 5A &#215; 104 &#215; 2.</li>
	</ol>
	</li>
	<li>Examination of LNC and BMMSC&#160;markers with flow cytometry15
	<ol>
		<li>Detect the expression of SCF, CD73, CD90, and CD105 in the LNCs and BMMSCs using Flow cytometry15 (2-4 &#215; 106 events/sample, 2,000 cells were obtained from each of the samples, totaling 550,000 cells that were gated simultaneously).</li>
		<li>Collect and stain the cells at 20-30 &#176;C for 15 min in a blocking buffer (3% BSA and 0.05% Tween-20 in PBS) using prelabeled antibodies (SCF [1:50], CD70 [1:50], CD90 [1:50], and CD105 [1:50]) as per the kit instructions.</li>
		<li>Perform fluorescence-activated cell sorting (FACS) analysis using a flow cytometer. In this study, FACS Diva software and FlowJo software were used. For each sample, record 10,000 events and gate and analyze live cells.</li>
	</ol>
	</li>
</ol>

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

<ol><li>Le, Q., Xu, J., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+diagnosis+of+limbal+stem+cell+deficiency%5BTitle%5D)+AND+%22Ocular+Surface%22%5BJournal%5D)">The diagnosis of limbal stem cell deficiency.</a> Ocular Surface. 16 (1), 58-69 (2018).</li>
<li>Polisetty, N., Fatima, A., Madhira, S. L., Sangwan, V. S., Vemuganti, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+cells+from+limbal+stroma+of+human+eye%5BTitle%5D)+AND+%22Molecular+Vision%22%5BJournal%5D)">Mesenchymal cells from limbal stroma of human eye.</a> Molecular Vision. 14, 431-442 (2008).</li>
<li>Xie, H. T., Chen, S. Y., Li, G. G., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+expansion+of+human+limbal+stromal+niche+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Isolation and expansion of human limbal stromal niche cells.</a> Investigative Ophthalmology & Visual Science. 53 (1), 279-286 (2012).</li>
<li>Li, G. G., Zhu, Y. T., Xie, H. T., Chen, S. Y., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+derived+from+human+limbal+niche+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Mesenchymal stem cells derived from human limbal niche cells.</a> Investigative Ophthalmology & Visual Science. 53 (9), 5686-5697 (2012).</li>
<li>Li, G. G., Chen, S. Y., Xie, H. T., Zhu, Y. T., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenesis+potential+of+human+limbal+stromal+niche+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Angiogenesis potential of human limbal stromal niche cells.</a> Investigative Ophthalmology & Visual Science. 53 (7), 3357-3367 (2012).</li>
<li>Hu, W., Zhang, Y., Tighe, S., Zhu, Y. T., Li, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+isolation+method+of+human+lacrimal+canaliculus+epithelial+stem+cells+by+maintaining+close+association+with+their+niche+cells%5BTitle%5D)+AND+%22International+Journal+of+Medical+Sciences%22%5BJournal%5D)">A new isolation method of human lacrimal canaliculus epithelial stem cells by maintaining close association with their niche cells.</a> International Journal of Medical Sciences. 15 (12), 1260-1267 (2018).</li>
<li>Kumar, A., Xu, Y., Yang, E., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stemness+and+regenerative+potential+of+corneal+stromal+stem+cells+and+their+secretome+after+long-term+storage%3A+Implications+for+ocular+regeneration%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Stemness and regenerative potential of corneal stromal stem cells and their secretome after long-term storage: Implications for ocular regeneration.</a> Investigative Ophthalmology & Visual Science. 59 (8), 3728-3738 (2018).</li>
<li>Xiao, Y. T., Qu, J. Y., Xie, H. T., Zhang, M. C., Zhao, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+methods+for+isolation+of+limbal+niche+cells%3A+Maintenance+of+limbal+epithelial+stem%2Fprogenitor+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">A comparison of methods for isolation of limbal niche cells: Maintenance of limbal epithelial stem/progenitor cells.</a> Investigative Ophthalmology & Visual Science. 61 (14), 16(2020).</li>
<li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limbal+niche+cells+and+three-dimensional+matrigel-induced+dedifferentiation+of+mature+corneal+epithelial+cells%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Limbal niche cells and three-dimensional matrigel-induced dedifferentiation of mature corneal epithelial cells.</a> Investigative Ophthalmology & Visual Science. 63 (5), 1(2022).</li>
<li>Basu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+limbal+biopsy-derived+stromal+stem+cells+prevent+corneal+scarring%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Human limbal biopsy-derived stromal stem cells prevent corneal scarring.</a> Science Translational Medicine. 6 (266), 266ra172(2014).</li>
<li>Acar, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+allogeneic+limbal+mesenchymal+stem+cell+therapy+in+corneal+healing%3A+role+of+administration+route%5BTitle%5D)+AND+%22Ophthalmic+Research%22%5BJournal%5D)">Effect of allogeneic limbal mesenchymal stem cell therapy in corneal healing: role of administration route.</a> Ophthalmic Research. 53 (2), 82-89 (2015).</li>
<li>Katikireddy, K. R., Dana, R., Jurkunas, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+potential+of+limbal+fibroblasts+and+bone+marrow+mesenchymal+stem+cells+to+corneal+epithelial+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.</a> Stem Cells. 32 (3), 717-729 (2014).</li>
<li>Polisetti, N., Sharaf, L., Reinhard, T., Schlunck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+ex+vivo+expansion+of+limbal+mesenchymal+stromal+cells%5BTitle%5D)+AND+%22Bio-Protocols%22%5BJournal%5D)">Isolation and ex vivo expansion of limbal mesenchymal stromal cells.</a> Bio-Protocols. 12 (14), e4471(2022).</li>
<li>Xie, H. T., Chen, S. Y., Li, G. G., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limbal+epithelial+stem%2Fprogenitor+cells+attract+stromal+niche+cells+by+SDF-1%2FCXCR4+signaling+to+prevent+differentiation%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation.</a> Stem Cells. 29 (11), 1874-1885 (2011).</li>
<li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+limbal+niche+cells+are+a+powerful+regenerative+source+for+the+prevention+of+limbal+stem+cell+deficiency+in+a+rabbit+model%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model.</a> Scientific Reports. 8, 6566(2018).</li>
<li>Kumar, A., Yun, H., Funderburgh, M. L., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+therapy+for+the+cornea%5BTitle%5D)+AND+%22Progress+In+Retinal+and+Eye+Research%22%5BJournal%5D)">Regenerative therapy for the cornea.</a> Progress In Retinal and Eye Research. 87, 101011(2022).</li>
<li>Pineda, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPineda%2C+R+%22World+Corneal+Blindness.+Foundations+of+Corneal+Disease%22">World Corneal Blindness. Foundations of Corneal Disease</a>. , Springer, Cham. 299-305 (2020).</li>
<li>Zieske, J. D., Guimarães, S. R., Hutcheon, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+keratocyte+proliferation+in+response+to+epithelial+debridement%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Kinetics of keratocyte proliferation in response to epithelial debridement.</a> Experimental Eye Research. 72 (1), 33-39 (2001).</li>
<li>Resnikoff, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+data+on+visual+impairment+in+the+year+2002%5BTitle%5D)+AND+%22Bulletin+of+the+World+Health+Organization%22%5BJournal%5D)">Global data on visual impairment in the year 2002.</a> Bulletin of the World Health Organization. 82 (11), 844-851 (2004).</li>
<li>Tan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limbal+bio-engineered+tissue+employing+3D+nanofiber-aerogel+scaffold+to+facilitate+LSCs+growth+and+migration%5BTitle%5D)+AND+%22Macromolecular+Bioscience%22%5BJournal%5D)">Limbal bio-engineered tissue employing 3D nanofiber-aerogel scaffold to facilitate LSCs growth and migration.</a> Macromolecular Bioscience. 22 (5), e2100441(2022).</li>
<li>Aghamirsalim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+printed+hydrogels+for+ocular+wound+healing%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">3D printed hydrogels for ocular wound healing.</a> Biomedicines. 10 (7), 1562(2022).</li>
<li>Sasamoto, Y., Ksander, B. R., Frank, M. H., Frank, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repairing+the+corneal+epithelium+using+limbal+stem+cells+or+alternative+cell-based+therapies%5BTitle%5D)+AND+%22Expert+Opinion+on+Biological+Therapy%22%5BJournal%5D)">Repairing the corneal epithelium using limbal stem cells or alternative cell-based therapies.</a> Expert Opinion on Biological Therapy. 18 (5), 505-513 (2018).</li>
<li>Rohaina, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstruction+of+limbal+stem+cell+deficient+corneal+surface+with+induced+human+bone+marrow+mesenchymal+stem+cells+on+amniotic+membrane%5BTitle%5D)+AND+%22Translational+Research%22%5BJournal%5D)">Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.</a> Translational Research. 163 (3), 200-210 (2014).</li>
<li>O'Callaghan, A. R., Dziasko, M. A., Sheth-Shah, R., Lewis, M. P., Daniels, J. T. J. A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+mucosa+tissue+equivalents+for+the+treatment+of+limbal+stem+cell+deficiency%5BTitle%5D)+AND+%22Advanced+Biosystems%22%5BJournal%5D)">Oral mucosa tissue equivalents for the treatment of limbal stem cell deficiency.</a> Advanced Biosystems. 4 (7), e1900265(2020).</li>
<li>Yu, D., Chen, M., Sun, X., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+mouse+induced+pluripotent+stem+cells+into+corneal+epithelial-like+cells%5BTitle%5D)+AND+%22Cell+Biology+International%22%5BJournal%5D)">Differentiation of mouse induced pluripotent stem cells into corneal epithelial-like cells.</a> Cell Biology International. 37 (1), 87-94 (2013).</li>
<li>Zeppieri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose-derived+stem+cells+for+corneal+wound+healing+after+laser-induced+corneal+lesions+in+mice%5BTitle%5D)+AND+%22Journal+of+Clinical+Medicine%22%5BJournal%5D)">Adipose-derived stem cells for corneal wound healing after laser-induced corneal lesions in mice.</a> Journal of Clinical Medicine. 6 (12), 115(2017).</li>
<li>Kumar, A., Kumar, V., Rattan, V., Jha, V., Bhattacharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secretome+cues+modulate+the+neurogenic+potential+of+bone+marrow+and+dental+stem+cells%5BTitle%5D)+AND+%22Molecular+Neurobiology%22%5BJournal%5D)">Secretome cues modulate the neurogenic potential of bone marrow and dental stem cells.</a> Molecular Neurobiology. 54 (6), 4672-4682 (2017).</li>
<li>Hayashi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordinated+generation+of+multiple+ocular-like+cell+lineages+and+fabrication+of+functional+corneal+epithelial+cell+sheets+from+human+iPS+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.</a> Nature Protocols. 12 (4), 683-696 (2017).</li>
<li>Guo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limbal+niche+cells+are+a+potent+resource+of+adult+mesenchymal+progenitors%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Limbal niche cells are a potent resource of adult mesenchymal progenitors.</a> Journal of Cellular and Molecular Medicine. 22 (7), 3315-3322 (2018).</li>
<li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+gene+expression+between+limbal+niche+progenitors+and+bone+marrow+derived+mesenchymal+stem+cells%5BTitle%5D)+AND+%22International+Journal+of+Medical+Sciences%22%5BJournal%5D)">Differential gene expression between limbal niche progenitors and bone marrow derived mesenchymal stem cells.</a> International Journal of Medical Sciences. 17 (4), 549-557 (2020).</li>
<li>González, S., Deng, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+native+limbal+stromal+cells+increases+the+expansion+efficiency+of+limbal+stem%2Fprogenitor+cells+in+culture%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture.</a> Experimental Eye Research. 116, 169-176 (2013).</li>
<li>Funderburgh, M. L., Du, Y., Mann, M. M., SundarRaj, N., Funderburgh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PAX6+expression+identifies+progenitor+cells+for+corneal+keratocytes%5BTitle%5D)+AND+%22FASEB+Journal%22%5BJournal%5D)">PAX6 expression identifies progenitor cells for corneal keratocytes.</a> FASEB Journal. 19 (10), 1371-1373 (2005).</li>
<li>Funderburgh, J. L., Funderburgh, M. L., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells+in+the+limbal+stroma%5BTitle%5D)+AND+%22Ocular+Surface%22%5BJournal%5D)">Stem cells in the limbal stroma.</a> Ocular Surface. 14 (2), 113-120 (2016).</li>
<li>Chen, S. Y., Hayashida, Y., Chen, M. Y., Xie, H. T., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+isolation+method+of+human+limbal+progenitor+cells+by+maintaining+close+association+with+their+niche+cells%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+C%2C+Methods%22%5BJournal%5D)">A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells.</a> Tissue Engineering. Part C, Methods. 17 (5), 537-548 (2011).</li>
<li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SnapShot%3A+Growing+organoids+from+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">SnapShot: Growing organoids from stem cells.</a> Cell. 161 (7), 1700-1701 (2015).</li>
</ol>

None of the authors have any relevant financial disclosures.

Corneal transparency is typically maintained by regular arrangement and distribution of small fibers (25-30 nm in diameter) in the corneal stroma, which is crucial for normal vision acuity16. There are 253 million visually impaired people worldwide, 36 million of whom are blind17. The world health organization (WHO) considers corneal blindness one of the most serious hazards to human eyesight, accounting for 5.1% of all blindness worldwide16. Corneal...

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.

<table><tbody><tr><td>4&#039;,6-Diamidino-2-Phenylindole</td><td>ThermoFisher</td><td>D1306</td><td>5&amp;mu;g/mL</td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>V900919</td><td>1.25 &amp;mu;g/mL</td></tr><tr><td>Anti-CD73</td><td>Abcam</td><td>ab202122</td><td>1:50</td></tr><tr><td>Bovine Serum Albumin</td><td>MERCK</td><td>A1933</td><td>-</td></tr><tr><td>CD105</td><td>Proteintech</td><td>67075-1-Ig</td><td>1:200</td></tr><tr><td>CD105</td><td>Abcam</td><td>ab114052</td><td>1:50</td></tr><tr><td>CD90</td><td>Proteintech</td><td>66766-1-Ig</td><td>1:100</td></tr><tr><td>CD90</td><td>Abcam</td><td>ab307736</td><td>1:50</td></tr><tr><td>Cell Incubator</td><td>Shanghai Lishen</td><td>K1119K4644</td><td>HF90(HT)</td></tr><tr><td>Centrifuge system</td><td>StatSpin&amp;nbsp;</td><td>StatSpin CytoFuge 12</td><td>-</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>2 mg/mL</td></tr><tr><td>Confocal microscope</td><td>Zeiss&amp;nbsp;</td><td>LSM700</td><td>-</td></tr><tr><td>Culture plate</td><td>virya</td><td>3500356</td><td>35 mm</td></tr><tr><td>DME/F-12 1:1 (1x)&amp;nbsp;</td><td>cytiva</td><td>SH30023.01</td><td>90%</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Secondary Antibody</td><td>ThermoFisher</td><td>A16016</td><td>1:1000</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Secondary Antibody</td><td>ThermoFisher</td><td>31568</td><td>1:1000</td></tr><tr><td>FACS Diva sofware</td><td>BD Biosciences</td><td>Tree Star</td><td>-</td></tr><tr><td>Flow Cytometer</td><td>BD Biosciences</td><td>Becton Dickinson LSRII</td><td>-</td></tr><tr><td>Fluorescence microscope</td><td>olympus</td><td>cx31&amp;nbsp;</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1914</td><td>50 &amp;mu;g/mL</td></tr><tr><td>Hemocytometer</td><td>MERCK</td><td>Z359629</td><td>Bright-Line</td></tr><tr><td>High-capacity cDNA Transcription Kit</td><td>ThermoFisher</td><td>4374966</td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td>&amp;nbsp;UOP</td><td>DSZ2000X</td><td></td></tr><tr><td>ITS (insulin, transferrin, sodium selenite)</td><td>Sigma</td><td>I3146</td><td>5 &amp;mu;g/mL insulin, 5 &amp;mu;g/mL transferrin, 5 ng/mL sodium selenite</td></tr><tr><td>KnockOut SR Serum Replacement for ESCs/iPSCs</td><td>gibco</td><td>10828-028</td><td>10%</td></tr><tr><td>Matrigel</td><td>BioCoat</td><td>356234</td><td>-</td></tr><tr><td>Pan-CK</td><td>Abcam</td><td>ab7753</td><td>1:1000</td></tr><tr><td>Paraformaldehyde</td><td>NoninBio</td><td>NBS0135</td><td>4.00%</td></tr><tr><td>Paraformaldehyde</td><td>MKBio</td><td>MM-1505</td><td>4%</td></tr><tr><td>PDGFR&amp;beta;</td><td>Abclonal</td><td>A1444</td><td>1:100</td></tr><tr><td>Real-time fluorescence quantitative PCR instrument</td><td>Applied Biosystems</td><td>Step One Plus</td><td>-</td></tr><tr><td>Recombinant Human FGF-basic</td><td>Peprotech</td><td>100-18B</td><td>4 ng/mL</td></tr><tr><td>Recominant Human Leukemia Inhibitory Factor(Lif)</td><td>Peprotech</td><td>300-05</td><td>10 ng/mL</td></tr><tr><td>RNeasy Mini RNA Isolation Kit</td><td>Qiagen</td><td>74104</td><td>-</td></tr><tr><td>SCF</td><td>Bioss</td><td>bs-0545R</td><td>1:100</td></tr><tr><td>SCF</td><td>Abcam</td><td>ab52603</td><td>1:50</td></tr><tr><td>Stereomicroscope</td><td>ZEISS</td><td>SteREO Discovery. V8</td><td></td></tr><tr><td>Sterile surgical round blade</td><td>Careforde</td><td>29500</td><td>size 10</td></tr><tr><td>TaqMan Gene Expression Assay Mix</td><td>Applied Biosystems</td><td>4448489</td><td></td></tr><tr><td>Triton X-100</td><td>MERCK</td><td>X100</td><td>0.20%</td></tr><tr><td>Trypan blue</td><td>ThermoFisher</td><td>15250061</td><td>0.40%</td></tr><tr><td>Trypsin-EDTA</td><td>Genview</td><td>GP3108</td><td>0.25%</td></tr><tr><td>Tween 20</td><td>MERCK</td><td>P9416</td><td>-</td></tr><tr><td>Ultra Clean Bench</td><td>LaiTe</td><td>LT20200705</td><td>SW-CJ-IFDG</td></tr><tr><td>Universal PCR Master Mix</td><td>Applied Biosystems</td><td>4304437</td><td></td></tr><tr><td>Vim&amp;nbsp;</td><td>Abcam</td><td>ab92547</td><td>1:100</td></tr></tbody></table>

isolation and identification of limbal niche cells

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 for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Growth of LNC 
The LNCs were successfully isolated according to the method of digestion of collagenase A (2 mg/mL) digestion of corneoscleral rim tissue, as described above (Figure 1). Consistent with a previously reported study3, after collagenase A digestion, caterpillar-like clusters were visualized under the microscope (Figure 2). The proportion of spindle cells increased gradually with the cell passage. Spindle-sh...

Here, we present a protocol to isolate and identify the human 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 ...

isolation-and-identification-of-limbal-niche-cells

Research

JoVE Journal

Medicine

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

The lacrimal gland (LG) has two cell types expressing α-smooth muscle actin (αSMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

Conjunctival Commensal Isolation and Identification in Mice

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Here, we present a protocol to obtain high-quality single-cell suspensions from whole mouse corneas through sequential digestion with collagenase A and trypsin. This method yields viable single cells with high RNA integrity, making them compatible with downstream applications such as single-cell RNA sequencing and flow cytometry.

A Protocol for Harvesting Single-cell Suspension from Mouse Corneas

Learn isolation and identification of human limbal niche cells for treating limbal stem cell deficiency and restoring vision. This protocol supports regenerative medicine research using single cell sequencing.

Isolation and Identification of Limbal Niche Cells

Explore More Videos