Isolation and Identification of Limbal Niche Cells

Summary

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

Abstract

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 °C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 °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 PDGFRβ). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFRβ, 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 68%, 99%, 20%, and 3% in BMMSCs. The standard process for LNC isolation and identification could provide a reliable laboratory basis for the widespread use of LNCs.

Introduction

The incidence of corneal epithelial stem cell deficiency (CESD), also called limbal stem cells deficiency (LSCD)¹, and corneal epithelial regeneration (CES) are becoming more and more urgent because of corneal infection and injury. If not properly treated, CESD can lead to blindness that requires corneal transplantation. As a result, CES regeneration is 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 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 and, with the function of bone marrow-derived MSC (BMMSCs), and could 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 "niche" 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 detailed procedure of LNC isolation, culture, and identification was recorded in the video for scientists who are interested in LNC study, and it can be conveniently repeated when needed.

Protocol

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

1. Obtain limbus tissue from intermediate-term corneal storage medium and operate under aseptic conditions on an ultra-clean workbench.
2. Scrape and remove the iris and endothelium around the cornea using a sterile surgical round blade.
3. Cut limbus tissue into twelve equally sized pieces with a surgical blade with 1 mm left on both sides of the cornea and sclera.
4. 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.
5. Put the plate into the 37 °C cell incubator, digest the limbal tissues for 18 h.
   NOTE: Follow-up procedures are usually performed on the second day.
6. Prepare a 5% basement membrane matrix (Matrigel) coated 6-well culture plate.
   1. Coat 5% basement membrane matrix (dissolved in MESCM [Table 1]) at the bottom of the well.
   2. Prepare 200 μL and 1 mL tips, one 15-mL centrifugal tube, and one 6-well plate in a sterilized bag.
   3. Put the sterilized bag (including tips and centrifugal tube) and a new 6-well plate at -20 °C or -80 °C environment for 20 min. Thaw the basement membrane matrix in the refrigerator at 4 °C.
   4. Take out the pre-cooled tips, centrifugal tube, and 6-well plate from the -20 °C or -80 °C environment, and then perform the following operations on the ultra-clean workbench.
   5. Transfer 50 µL 5% basement membrane matrix to 1 mL MESCM using a 200 μL tip, and mix them gently and evenly.
   6. Transfer 5% basement membrane matrix (dissolved in MESCM) to one well of a 6-well culture plate using the pre-cooled 1 mL tip.
   7. Shake the 6-well plate gently and horizontally, put the 6-well culture plate in the 37 °C cell incubator for approximately 1 h, and then perform the next step.
7. After 18-h digestion, take out collagenase A-digested limbal tissue, 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).
8. Use a 200 µL pipette tip to detach the clusters from undigested scleral tissues under the stereomicroscope.
9. 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.
10. 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.
11. Transfer the cell suspension to a 15-mL centrifugal tube and centrifuge at 200 x g for 5 min.
12. Remove the supernatant carefully without disturbing the cell precipitation, and then add 1 mL MESCM to resuspend the cells.
13. 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 µL cell suspension for cell counting.
14. 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.
15. Gently shake the 6-well plate horizontally to evenly distribute the cells.
16. 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.

2. Identification of LNC

1. Immunofluorescence staining
   1. Digest the LNCs from the bottom of the plate using 1 mL 0.25% TE for 5 min in the 37°C cell incubator.
   2. 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.
   3. Add 1 mL MESCM to resuspend the cells in suspension. Prepare 80 µL cell suspension (4 × 10³ cells per slide) for 4 slides (20 µL/slide) and use centrifuge system to deposit cells onto microscope slides as per the manufacturer's instructions.
   4. 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.
   5. 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], PDGFRβ [1:100]) on a shaker overnight at 4 °C. After the incubation, remove unbound primary antibodies by washing (5 min/wash, 3x) the slides with PBST (PBS + 0.1% Tween 20).
   6. 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).
   7. Counterstain the nuclei with 5 µg/mL DAPI (4',6-Diamidino-2-Phenylindole ) and seal the slides.
   8. Perform fluorescence imaging using a fluorescence microscope (Magnification: 400x; Exposure time: 50 ms).
2. Quantitative real-time polymerase chain reaction (qPCR)
   1. Take RNA Isolation Kit to extract the total RNA as per the manufacturer's instructions.
   2. Use a high-capacity cDNA Transcription Kit to reverse transcribe 1-2 µg of total RNA to cDNA.
   3. Perform the reverse transcription (42 °C, 2 min; 50 °C , 85 min) and qPCR ( 95 °C, 5 min; 95 °C, 10 s, 40 cycles) in a 20-μL solution containing cDNA (100 ng), TaqMan Gene Expression Assay Mix (1 μL), universal PCR Master Mix (10 μL) and ddH₂O (to 20 µL).
   4. Use the comparative CT technique (ΔΔCT)^4,9 to examine the relative gene expression.

3. Characterization of LNC

1. Cell counting (hemocytometer)
   1. Digest the LNCs from the bottom of the plate using 0.25% TE for 5 min in the 37 °C cell incubator.
   2. 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.
   3. Add 1 mL MESCM to resuspend the cells, then take 10 μL cell suspension in a 0.5 mL centrifuge tube. Add an equal volume of 0.4% Trypan blue staining solution.
   4. Place 10 μL 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 "A"; the cell number in 1 mL is calculated as 5A × 10⁴ × 2.
2. Examination of LNC and BMMSC markers with flow cytometry¹⁵
   1. Detect the expression of SCF, CD73, CD90, and CD105 in the LNCs and BMMSCs using Flow cytometry¹⁵ (2-4 × 10⁶ events/sample, 2,000 cells were obtained from each of the samples, totaling 550,000 cells that were gated simultaneously).
   2. Collect and stain the cells at 20-30 °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.
   3. 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.

Results

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. Spi...

Discussion

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...

Materials

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