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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#39;,6-Diamidino-2-Phenylindole</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>5&#956;g/mL</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>V900919</td>
			<td>1.25 &#956;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&#160;</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&#160;</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)&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1914</td>
			<td>50 &#956;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>&#160;UOP</td>
			<td>DSZ2000X</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS (insulin, transferrin, sodium selenite)</td>
			<td>Sigma</td>
			<td>I3146</td>
			<td>5 &#956;g/mL insulin, 5 &#956;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&#946;</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&#160;</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

902 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 

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

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 ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...