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

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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

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&#160;cell passage. Spi...

Watch this Scientific Journal Video about Standardizing Limbal Niche Cell LNC Isolation and Characterization to Support Widespread LNC Research at JoVE.com

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

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

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

Research

JoVE Journal

Medicine

924 Views.  Huazhong University of Science and Technology. Here, we present a protocol to isolate and identify the human limbal niche cells.

Video: Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

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

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

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.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Developmental Biology

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 goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

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.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

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

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

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.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

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

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

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.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

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

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

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.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Immunology and Infection

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

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

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.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

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.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

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

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

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.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

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

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

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.

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...