CN111575234A - Separation culture method of human cornea Niche cells - Google Patents

Abstract

The invention discloses a separation culture method of human cornea Niche cells. The method comprises removing inner cortex of corneal flap left after corneal transplantation, and digesting with Dispase solution in 37 deg.C water bath; then EDTA solution is used for incubation at room temperature to obtain cell suspension; adding a KCM culture solution into the cell suspension for culturing; and when the ready-made fibroid cells appear around the corneal epithelial clone, scraping the epithelial cells to selectively retain the fibroid cells, thus obtaining the primary corneal Niche cells. The invention separates the cornea Niche cells (LNCs) from the corneal limbus by a digestion method, which greatly facilitates the separation and culture of the cornea Niche cells.

Description

Separation culture method of human cornea Niche cells

Technical Field

The invention relates to the field of corneal cells, in particular to a separation culture method of human corneal Niche cells.

Background

Numerous studies have shown that the microenvironment plays a crucial role in maintaining and activating human stem cell activity, such as small intestine stem cells, hair follicle stem cells, and neural stem cells. Stem cell Niche is a special microenvironment consisting of cells and Extracellular components, including a special Extracellular Matrix (ECM) and secreted cytokines. The interaction of peripheral cells, extracellular matrix and soluble signaling mediators has an important role in maintaining stem cell stability and activation. The understanding and understanding of the stem cell Niche play an important role in the research of the Niche regulation of the self-renewal of the stem cells, the function of the stem cells and the application of the stem cells in clinical regenerative medicine.

Limbal stem cells are generally accepted to be distributed in the Vogt palisade of the limbus and the Niche chamber of its adjacent tissues, maintaining the renewal of corneal epithelial cells. Limbal stem cells, located in the basal layer of the limbus, have slow-cycle cellular characteristics, are highly proliferative, and express several recognized corneal epithelial stem cell markers, such as Δ Np63 α, ABCG2,

β 1-intergrin and N-cadherin; but does not express corneal epithelial differentiation markers such as keratin K3/12, connexin 43. Cells adjacent to stem cell Niche include melanocytes, antigen presenting Langerhans cells, suppressor T lymphocytes and recently discovered corneal Niche cells. The anatomical structure of the Niche chamber breaks through Bowman's membrane and extends into the limbal stromal layer rich in cells and blood vessels, indicating that the limbal stem cells are closely related to the peripheral limbal stromal cells.

The corneal limbal Niche cell dissection part is adjacent to the corneal limbal basal cell, and the Niche cell is separated and found to have the characteristic of maintaining the growth of the corneal limbal stem cell through a stromal derived factor SDF-1 and a receptor CXCR4(SDF-1-CXCR4) signal path. It has also been shown that stromal cell LSCs located in the limbal stroma can also support the proliferation of limbal stem cells, and even induce differentiation of hair follicle stem cells and embryonic stem cells into corneal epithelial-like cells, and these results indicate that LSCs may have an important role in the limbal stem cell microenvironment. Because the limbal Niche cell LNCs and the limbal stromal cell LSCs are in close anatomical proximity and both have the property of supporting the growth of limbal stem cells, they are often confused by scholars in the study. The method has great significance for further researching and understanding the structure and the function of the limbal stem cell Niche. However, no study has been conducted specifically on these two cells.

Therefore, there is a need in the market for a method for isolated culture of human corneal Niche cells that can solve the above problems.

Disclosure of Invention

In order to solve one or more problems in the prior art, the invention provides a separation culture method of human cornea Niche cells.

The technical scheme adopted by the invention to achieve the aim is as follows: a method for isolated culture of human corneal Niche cells, the method comprising:

tearing off the endothelial layer of the corneal flap left after the plating layer cornea transplantation, and digesting the corneal flap with Dispase solution in water bath at 37 ℃;

then EDTA solution is used for incubation at room temperature to obtain cell suspension;

adding a KCM culture solution into the cell suspension for culturing;

and when the ready-made fibroid cells appear around the corneal epithelial clone, scraping the epithelial cells to selectively retain the fibroid cells, thus obtaining the primary corneal Niche cells.

In some embodiments, the enzyme activity in the Dispase solution is 2.4U/ml.

In some embodiments, the time for digestion in the water bath is 1 hour.

In some embodiments, the concentration of the EDTA solution is 0.02%.

In some embodiments, the incubation time is 2 minutes.

The invention has the beneficial effects that: the invention separates cornea Niche cells (LNCs) from the corneal limbus by a digestion method, separates corneal Limbus Stromal Cells (LSCs) from the corneal limbus stroma by a tissue mass culture method, and cultures and subcultures the separated cells under the same conditions. LNCs and LSCs were co-cultured with limbal stem cells as feeder cells after treatment with mitomycin c (mmc), respectively, and the colony formation rates (CFE), epithelial cell repolarization, and expression of limbal stem cell markers were compared between the two groups of limbal stem cells. The gene expression of the growth factors supporting the growth of the limbal stem cells of the two groups of cells is compared, and the comparison shows that the LNCs can better support the growth of the limbal stem cells and maintain the characteristics of the stem cells than the LSCs, and the two cells play different roles in maintaining normal ocular surface epithelium; the comparison method can accurately and quickly carry out comprehensive analysis on the functions of the Niche cells and the limbal stromal cells for maintaining the limbal stem cells.

Drawings

FIG. 1 is a diagram illustrating the identification of limbal stem cells, Niche cells and stromal cells in a method for isolated culture of human corneal Niche cells according to a preferred embodiment of the invention;

FIG. 2 is a diagram showing the identification of limbal stem cells and Niche cells in a method for isolated culture of human corneal Niche cells according to a preferred embodiment of the present invention;

FIG. 3 is a diagram showing the coculture of limbal mixed cell fluid and feeder cells collected from the separation and culture of LNCs and LSCs in a method for separating and culturing human corneal Niche cells according to a preferred embodiment of the present invention;

FIG. 4 is a diagram of the formation of epithelial clones after 10-13 days of coculture of primary limbal stem cells and LNCs, LSCs and NIH/3T3 cells in a colony formation experiment in a method for isolated culture of human corneal Niche cells according to a preferred embodiment of the present invention;

FIG. 5 is a diagram illustrating the clonal feature of limbal stem cells in a method for isolated culture of human corneal Niche cells in accordance with a preferred embodiment of the present invention;

FIG. 6 is a diagram showing the characteristic of corneal epithelial cells cultured in vitro in the method for isolated culture of human corneal Niche cells according to the preferred embodiment of the present invention;

FIG. 7 is a diagram showing different cell phenotypes of LNCs and LSCs in a method for isolated culture of human corneal Niche cells according to a preferred embodiment of the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

FIG. 1 identification of limbal stem cells, Niche cells, stromal cells; normal limbal tissue remains multicellular in typical stratified epithelium and its underlying stromal cell layer (a), p63+ limbal stem cells and vimentin + limbal mesenchymal cells (arrows) are co-distributed within the limbal epithelial basal lamina crypt, and the inserted white boxed area is shown enlarged in the lower panel (B). After digestion with neutral protease, the limbal epithelial layer is loosened from the stromal layer (C), the loosened epithelial layer contains p63+ limbal stem cells and vimentin + mesenchymal cells (arrows) (D), and the loosened epithelial cells are mechanically separated with a cell scraper. After enzymatic digestion and mechanical separation, only very small amounts of p63+ epithelial cells and vimentin + mesenchymal cells remained in the basal layer of the limbal tissue (E, F). A scale: 100 μm, 300 μm. Vim, vimentin.

FIG. 2 identification of limbal stem cells and Niche cells; the collected limbal mixed cell suspension is prepared into a cell flail through centrifugation, and the result of immunofluorescence staining shows that the p63+ limbal stem cells, the vimentin + and the p 63-mesenchymal cells are collected together after enzyme digestion and mechanical separation.

FIG. 3 isolation and culture of LNCs and LSCs; the collected limbal mixed cell fluid was not co-cultured with feeder cells, cultured in KCM medium for 10-14 days, fibroblast-like mesenchymal cells (defined as LNCs) were grown around epithelial clones (A), and digested for passage after scraping corneal epithelial cells (C). Immunohistochemical staining showed that K3 positive epithelial cell clones were surrounded by vimentin + fibroblast-like cells (E). LSCs crawled out of corneal stromal tissue patch cultures on days 3-5 (B), and after passage (D) immunohistochemical staining revealed vimentin + fibroblasts (F). A scale: 200 μm. Vim, vimentin.

FIG. 4 clone formation experiments; primary limbal stem cells were formed as epithelial clones after 10-13 days of co-culture with LNCs, LSCs and NIH/3T3 cells. The group of LSCs formed fewer epithelial clones than the group of LNCs and NIH/3T3 (A). The NIH/3T3 group clones showed more pronounced epithelial cell differentiation morphology compared to LNCs, whereas the LSCs group formed clones with smaller cell numbers, ruffled edges, highly irregular shapes (B). Quantitative analysis of colony formation showed that colony formation rates CFE were significantly higher in the LNCs group and the NIH/3T3 group than in the LSCs group (C). A scale: 200 μm. P < 0.05.

FIG. 5 limbal stem cell clonal signature; immunohistochemistry and mRNA quantitative analysis results show that the NIH/3T3 group highly expresses a corneal epithelial differentiation marker K3(A, C), and the LNCs group highly expresses a limbal stem cell marker delta Np63(B, D). A scale: 200 μm. P < 0.05.

FIG. 6 characteristics of corneal epithelial cell sheets cultured in vitro; after 3 weeks of in vitro culture of LNCs, LSCs and NIH/3T3, three groups all formed a stratified corneal epithelial cell sheet. The limbal stem cells from the group of NIH/3T3 and LNCs formed 4-5 stratified epithelial sheets, whereas the corneal epithelial cells from the group of LSCs formed only 2-3 layers (A). The epithelial cell sheet was subjected to immunofluorescence K3 staining (B), Δ Np63 staining (C) and K12 staining (D). The results of the semi-quantitative analysis of fluorescence intensity show that: the corneal specific marker K12 expression level of the corneal epithelium of three groups has no obvious difference, but the NIH/3T3 group has higher expression of the corneal epithelium differentiation marker K3 (E). The percentage of Δ Np63+ cells in the group of LNCs was significantly higher than in the group of LSCs and NIH/3T3 (F). A scale: 200 μm. P < 0.05.

FIG. 7 analysis of cellular genes of LNCs and LSCs for different cell phenotypes LNCs and LSCs demonstrated expression of vimentin, and no expression of K3 and K4, excluding corneal epithelial cell contamination (A). Both cells expressed the same levels of EGF, FGF2, EPR, HGF, KGF, NGF, GDNF, BDNF, N-cadherin and Import 13. However, LNCs expressed low NT3 but significantly higher E-cadherin (b) than LSCs. Vim, vimentin; k3, cytokeratin 3; k4, cytokeratin 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NT, no reverse transcriptase; NT3, Neurotrophin-3; e-cad, E-cadherin; EGF, epiermal fibroplast growth factor; FGF2, fibroblast growth factor 2; EPR, epiregulin; HGF, hepatocyte growth factor; KGF, keratinocyte growth factor; NGF, new growth factor; GDNF, global cell-derived neurotropic factor; BDNF, blue-derived neuroviral factor; n-cad, N-cadherin; IPO13, import in 13.

Referring to fig. 1-7, the invention discloses a separation culture method of human cornea Niche cells, which is characterized in that the method comprises the following steps:

step 01, separating and culturing limbal stem cells, corneal Niche cells and limbal stromal cells;

the corneal donors used in the experiments (n ═ 6) were all obtained from the northern beans (Seattle, WA) eye bank. Comparison between LNCs and LSCs cells was performed between cells isolated from the same piece of cornea, and the results were averaged over three experiments. The corneal lamellae remaining after the lamellar keratoplasty were removed and digested with 2.4U/ml Dispase solution (BDbiosciences, Bedford, Mass.) in a 37 ℃ water bath for 1 hour, followed by incubation with 0.02% EDTA solution (Nacalai Tesque, Kyoto, Japan) for 2 minutes at room temperature. Epithelial cells were scraped along the limbus with sterile surgical forceps and the collected cells were incubated with 0.25% pancreatin-ethylenediaminetetraacetic acid (EDTA) (Nacalai Tesque, Kyoto, Japan) for 15 minutes at 37 ℃. The resulting cell suspension is cultured with a modified Keratinocyte Culture Medium (KCM) to obtain primary limbal stem cells. The improved keratinocyte cell culture medium comprises the following components: DMEM medium (Dulbecco 'digested eagle medium) and Ham's F12 medium (DMEM/F12,3:1), 5% Fetal Bovine Serum (FBS) (Invitrogen),1nM cholera toxin (Calbiochem, La Jolla, CA),2nM triiodothyronine (Takeda, Osaka, Japan), 0.4. mu.g/ml hydrocortisone (Kowa, Tokyo, Japan), 1% insulin-transferrin selenium supplement (Invitrogen),10ng/ml human recombinant epidermal growth factor (Sigma, St. Louis, MO)100U/ml penicillin, 100. mu.g/ml streptomycin (Invitrogen). Adding KCM culture solution into the collected cell suspension, culturing for 10-14 days, scraping epithelial cells to selectively retain fibroblast when the fibroblast appears around the corneal epithelial clone, and obtaining the primary corneal Niche cells (LNCs).

And (3) LSCs cell culture: after removing the anterior stroma layer (containing residual epithelial cells) and the posterior stroma layer (containing corneal endothelial cells) of the corneal limbus tissue, the corneal limbus tissue is paved in a 10% FBS-DMEM medium for culture until the corneal Limbus Stromal Cells (LSCs) proliferate and climb out of the tissue. The LNCs and LSCs were cultured in 10% FBS-DMEM medium in 5% CO2 at 37 ℃ incubator with medium changes every 3 days.

Step 02, preparing a feeder cell layer;

to compare the ability of different feeder cells to support the proliferation of limbal stem cells, three cells of NIH/3T3, LNCs, LSCs (LNCs and LSCs are second generation cells) were cultured in medium supplemented with Mitomycin C (MMC) ((KyowaHakko, Tokyo, Japan) at 37 ℃ for 2 hours to remove mitotic activity, 2X 104 cells/cm 2, 1.5X 104 cells/cm 2, 1.0X 104 cells/cm 2, 0.8X 104 cells/cm 2 and 0.5X 104 cells/cm 2 cell concentration of LNCs and LSCs feeder cells, respectively, and after experimental comparison, the colony formation rate CFE was found to be highest for the group of feeder cells seeded at a cell density of 0.8X 104 cells/cm 2, and therefore we selected this concentration for the subsequent experiments for 3 washes, after trypsinization, and seeded with Kigelwell cells at a density of 0.8X 104 cells/cm 2 (Gelsc) in Gelatil wells (3623 cells) or in gell 366 wells, nitta Gelatin, Osaka, Japan) as feeder cell layer.

Step 03, cloning and forming experiments;

inoculating primary limbal stem cells into a 6-well plate covered with TERT + TK-D or 3T3 feeder cell layer at a density of 1000 per well, culturing for 10-13 days, fixing with 10% neutral formalin, staining with 1% rhodamine B (Wako, Osaka, Japan), and cloning efficiency (CFE%) -clone number/inoculated viable cell number × 100%;

step 04, preparing a corneal epithelial cell sheet;

inoculating 1-2 × 105 primary limbal stem cells per well into a Transwell chamber containing a collagen gel coating of feeder cell layer; after the cells are cultured in a KCM culture medium for 12 days, reducing the liquid level culture for 6 days to promote corneal epithelium stratification; changing the liquid every 2 days;

step 05, reverse transcription PCR and quantitative PCR;

total RNA was extracted from each tissue and cell using the RNeasy Mini Kit (Qiagen, Valencia, Calif.) with instructions. The cDNA required for PCR was synthesized by reverse transcription and translation using 1st Strand cDNA Synthesis System (origin, Rockville, Md.). PCR thermal cycling stripThe parts are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, extension at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 10min at 72 ℃. And (3) identifying the PCR product by using 2% agarose gel electrophoresis, observing by using an ultraviolet projection imaging system, and analyzing the relative content of the target gene by using gel image analysis software. The sequence information of the PCR specific primers is shown in Table 1. The expression of each gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Quantitative PCR use

The fluorescent quantitative PCR method is carried out by detecting and analyzing using an Applied Biosystems 7900HT (Applied Biosystems, Foster City, Calif.) sequence detector. Δ Np63(Hs00978339_ m1), keratin 3(K3) (Hs00365080_ m1), GAPDH (Hs99999905_ m1) sequences were purchased from applied biosystems. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 sec; denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 1min, for 45 cycles. The quantitative data were normalized using GAPDH as an internal reference and analyzed using sequence detection system software (applied biosystems). The final results were averaged over the three experimental data.

Step 06, immunohistochemical staining;

the collected limbal cell suspension was loaded into a 6.0X 104 cell/tube, centrifuged at 1500rpm for 5min by a Cytofug centrifuge (StatSpin, Inc.), prepared into a cell slide, dried at room temperature for 5min, fixed with 4% paraformaldehyde for 15min, and then subjected to immunofluorescence staining.

Untreated, neutral protease-treated, and mechanically isolated corneal donor tissue, as well as harvested corneal epithelial regenerative cell sheets, were embedded, frozen sections of 6-8 μm thickness were performed, and then fixed with 4% paraformaldehyde at 4 ℃ for 30min for immunofluorescence staining.

Immunofluorescence staining procedure: sections were fixed with 4% paraformaldehyde at 4 ℃ for 30min, 4% skim milk in PBS (containing 0.3% Triton X-100) for blocking nonspecific binding at room temperature for 1h, after which primary antibody: anti-K3(AE5) (1:100, Progen Biotechnology, Heidelberg, Germany), anti-keratin12(1:100, Santa Cruz Biotechnology, Santa Cruz, Calif.), and anti-P63(4A4) (1:100, Santa Cruz), incubated overnight at 4 ℃. Then washed 3 times with PBS for 5min each time, and added with corresponding secondary antibody for incubation for 1h at normal temperature. The nuclei were washed 3 times with PBS and finally stained with DAPI and observed with a Zeiss fluorescent microscope (Axiovert 200M, Carl Zeiss Jena Gmbh, Jena Germany). Sections of the same concentration of non-specific antibody were added as negative controls. H & E staining was performed according to conventional methods. The immunofluorescent staining image signals of the epithelial cell sheets were quantified using NIH ImageJ software. One sample is quantified by selecting three different immunofluorescence photographs, and the number of cell nuclei is counted. Relative fluorescence intensity is total fluorescence intensity/total number of cells. The average of the three data sets was taken.

The data obtained above were all processed with SPSS 16.0 statistical analysis software (SPSS inc., Chicago, IL, USA). Data were analyzed using single sample analysis of variance (One-Way ANOVA) with significance for differences with p < 0.05.

Step 07, identifying limbal stem cells, Niche cells and stromal cells;

limbus analysis was first performed by H & E staining of limbal tissue

Organization of Niche. The results show that the limbal tissue has a typical stratified epithelial cell layer, with a high cell density limbal stromal region below (FIG. 1A). Limbal stem cells (fig. 1B, p63+ cells) were distributed in the basal layer of the limbal epithelium, while Vimentin-positive (Vimentin +) mesenchymal cells were distributed in the limbal epithelial crypts (arrows) near the basal epithelium (fig. 1B). However, after neutral protease treatment, the limbal epithelium loosened and left the stromal layer (fig. 1C). The loose limbal cell sheet contained not only P63+ epithelial cells but also vimentin +, P63-cells (arrows) (fig. 1D). Cells above the dashed line (P63+ limbal stem cells and vimentin +, P63-corneal Niche cells) were mechanically isolated for culturing LNCs (FIG. 1C, D). P63+ limbal stem cells and vicinal vimentin +, P63-corneal Niche cells were not present in the limbal tissue remaining after mechanical scraping, whereas some vimentin + stromal cells were still present in the deep layers of the remaining stromal layer (fig. 1E, F). In addition, we confirmed that limbal stem cells and corneal Niche cells coexist in the collected mixed cell sap by immunofluorescence staining of the limbal mixed cell sap centrifugal flap. Immunofluorescent staining showed that P63+ limbal stem cells and vimentin +, P63-corneal Niche cells were collected by digestion and mechanical separation (fig. 2).

Step 08, separating and culturing LNCs and LSCs;

the limbal cell mixture collected by digestion and mechanical separation was plated onto culture plates and cultured in feeder cells-free, KCM medium to obtain LNCs. On day 7 of culture, a typical corneal epithelial cell clone began to form, and by day 10-14, many fibroblast-like mesenchymal cells appeared around the clone (fig. 3A). Fibroblast-like cells (LNCs for short) were detached by trypsinization after mechanical scraping of epithelial cell clones (fig. 3C). LSCs are cultured from tissue pieces remaining after digestion and separation of the epithelial layer. To prevent the incorporation of epithelial cells into the Niche cells, the experiment used only the central portion of the corneal stroma layer. Corneal stromal cells migrated from the tissue 3-5 days later (FIG. 3B), and subcultured for subsequent experiments (FIG. 3D). Furthermore, immunofluorescent staining results showed that fibroblast-like mesenchymal cells were surrounded by epithelial cell clones (keratin K3+) when LNCs were cultured (vimentin +) (fig. 3E), whereas only vimentin + fibroblasts were observed when LSCs were cultured (fig. 3F).

Step 09, cloning formation and stem cell characteristics;

the limbal stem cells were co-cultured with three feeder cells, LNCs, LSCs, NIH/3T3, to compare the differences in the ability of the three to maintain limbal stem cell growth. The results showed that at days 10-13, three groups of cells formed typical epithelial cell clones; however, the colony formation rate CFE of the group of LSCs was lower than that of the group of LNCs and NIH/3T3 (FIG. 4A). Compared with the LNCs group, the NIH/3T3 group formed cell clones exhibiting highly differentiated epithelial cell morphology, whereas the LSCs group formed small colonies with a low number of cells, ruffled edges and highly irregular shapes (fig. 4B). Quantitative analysis of colony formation showed that the colony formation rate CFE was significantly higher for the LNCs group (CFE, 6.57. + -. 1.54%) and the NIH/3T3 group (CFE, 10.57. + -. 1.46%) than the LSCs group (CFE, 1.43. + -. 0.47%) (FIG. 4C). In addition, immunofluorescence staining and mRNA quantitation showed that the NIH/3T3 group highly expressed the corneal epithelial cell differentiation marker K3 (fig. 5A, C), while the LNCs group highly expressed the limbal stem cell marker Δ Np63 (fig. 5B, D).

Step 10, characteristics of the cultured corneal epithelial cell sheet;

after 3 weeks of co-culture, three groups of LNCs, LSCs and NIH/3T3 all form a stratified corneal epithelial cell sheet. Morphological observation showed that the limbal stem cells from the group of NIH/3T3 and LNCs formed 4-5 layers of stratified epithelial cells, whereas the corneal epithelial cells from the group of LSCs formed only 2-3 layers (FIG. 6A). Immunofluorescence staining results show that the NIH/3T3 group corneal epithelial cell sheet expresses high K3 positive staining (fig. 6B, E), and the limbal stem cell marker Δ Np63 stains only in basal cells, while the LNCs group epithelial cell sheet highly expresses Δ Np63 in both basal and pteroid cells (fig. 6C, F). There were no significant differences in the staining of the three groups of co-cultured epithelial cytokeratin K12 (fig. 6D, E).

And step 11, obtaining the advantages and disadvantages of the LNCs and the LSCs for maintaining the functions of the limbal stem cells through the phenotypic characteristics of the LNCs and the LSCs.

Reverse transcription PCR analysis showed that LNCs and LSCs cultured in vitro expressed Vimentin, but did not express the corneal epithelial cell marker keratin K3 and the conjunctival cell marker keratin K4, indicating that these two cells were not contaminated by corneal epithelial cells and conjunctival cells (fig. 7A). We also refer to documents 23-26, where genes involved in supporting the growth of limbal stem cells were selected and tested for expression in LNCs and LSCs. The results showed that both LNCs and LSCs expressed epidermal fibroblast growth factor (EGF), basic fibroblast growth factor 2(FGF2), epidermal regulator (EPR), Hepatocyte Growth Factor (HGF), Keratinocyte Growth Factor (KGF), Nerve Growth Factor (NGF), glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), N-cadherin, and import 13. However, LNCs expressed neurotrophin 3(NT3) less than LSCs, with significantly higher expression of E-cadherin (fig. 7B).

In this study, we report a new method for isolating limbal Niche cells and compare the difference between limbal Niche cells and limbal stromal cells in maintaining the growth of limbal stem cells. The results show that LNCs have a greater ability to maintain limbal stem cell growth than LSCs, including expression of limbal stem cell markers, colony formation, and regeneration of stratified corneal epithelium. Furthermore, LNCs, in comparison with LSCs, express E-cadherin at a high level but NT3 at a low level, in addition to conventional growth-related factors such as EGF, FGF2, EPR, HGF, KGF, NGF, GDNF, BDNF, N-cadherin, and Import 13, and the like. Limbal stem cells are generally accepted to be distributed within the limbal Vogt-fence-like structure. Cells within the corneal stroma beneath the basement membrane of the limbal epithelial cells have heterogeneity and have been poorly studied. Recently, Scheffer et al discovered that a new cell is located in the basal layer of the limbal epithelium and has the property of maintaining limbal stem cells, which are called limbal Niche cells. We also found in the experiment of culturing corneal limbal stem cells that the corneal tissue treated by neutral protease is separated by strong mechanical force to obtain more clones. Studies also demonstrated that very few vimentin + cells remained in the basal cell layer after enzymatic digestion and mechanical separation, indicating that mechanical separation separates basal epithelial cells from Niche cells more than enzymatic digestion alone. In fact, immunofluorescence staining of the limbal mixed cell fluid centrifugal spinning confirmed that both the p63+ limbal stem cells and adjacent vimentin + mesenchymal cells were collected together after enzymatic digestion and mechanical separation. In previous experiments, we observed that after the limbal stem cells are cultured in KCM culture solution alone for a period of time, there is proliferation of vimentin + fibroblast-like mesenchymal cells around corneal epithelial clones. Given the interplay between limbal cells, these fibroblast-like cells grew out around epithelial clones, most likely limbal Niche cells LNCs. Furthermore, although LNCs and LSCs cells have similar cell morphology and genetic phenotype, we only chose the intermediate layer of the limbal stroma to perform experiments in order to avoid contamination of the Niche cells. It is therefore possible to remove some of the most potent stromal cells adjacent to the basal cell layer, thereby reducing the limbal stromal cell's ability to support limbal stem cells. Meanwhile, the culture medium used in the experiment contains fetal calf serum, and the serum can promote the cell differentiation of LNCs and LSCs, so that only the second generation cells are selected as feeder cells for research in the experiment. Our studies, as with previous studies, show that LNCs have a greater ability to maintain limbal stem cell growth than LSCs, and that epithelial cells can be maintained to express more stem cell markers, form more epithelial clones, and form stratified corneal epithelial sheets. The results of the study showed that the group of LNCs formed 4-5 sheets of stratified corneal epithelial cells, whereas the group of LSCs formed only 2-3 sheets of stratified cells. Interestingly, the LNCs group cell sheet expressed the corneal epithelial differentiation marker K3 mainly in the superficial cell layer. Furthermore, compared with the NHI/3T3 group, the epithelial cells collected in the colony formation experiment and the cell sheet culture experiment showed lower expression of K3. More particularly, immunohistochemical staining and mRNA quantification experiments of epithelial clones showed that the group of LNCs expressed more Δ Np63+ cells in the basal and pterygoid cell layers than the NHI/3T3 group, indicating that LNCs were more effective in maintaining the stem cell characteristics of limbal stem cells than LSCs and NHI/3T3 cells. Therefore, it is believed that LNCs are effective in supporting the proliferation of limbal stem cells, while maintaining the undifferentiated properties of limbal stem cells in vitro. Experiments LNCs and LSCs were cultured in 10% FBS-DMEM media, which was suitable for fibroblast growth and not suitable for corneal epithelial cell growth. The isolated cells showed a fibroblast-like morphology, negative expression of the corneal epithelial differentiation marker K3, conjunctival epithelial marker K4, ensuring that LNCs and LSCs are not contaminated by corneal and conjunctival epithelial cells. In addition, reverse transcription PCR results showed that both LNCs and LSCs expressed multiple growth factors (EGF, FGF2, EPR, HGF, KGF, NGF, GDNF, and BDNF) and the potential corneal epithelial stem cell markers N-cadherin and import 13. However, LNCs express E-cadherin more strongly and NT3 less strongly. Growth factors play an important role in both corneal epithelium renewal and injury repair, with most of them being stimulated by fibroblast cells to proliferate and differentiate by acting on epithelial cell receptors by a paracrine process. Our studies demonstrated that both LSCs and LNCs secreted multiple growth factors. Although LSCs express NT3 more than LNCs, NT3 does not work strongly on limbal stem cells because of less expression of the NT3 receptor TrkC on limbal stem cells, consistent with our findings. Cadherins mediate cell-to-cell adhesion, play an important role in embryonic development, and play an important role in tissue formation, cell growth and differentiation. Research finds that N-cadherin (neural cadherin) plays an important role in the interaction between hematopoietic stem/progenitor cells and limbal stem cells and Niche cells. The limbal stem cells secrete N-cadherin factors like a molecular anchor, and the limbal stem cells are anchored and adhered to N-cadherin + expressed Niche cells. LNCs are distributed in the basal layer of the limbal epithelium, express N-cadherin proteins, which may act directly on limbal stem cells by anchoring to N-cadherin and E-cadherin (epithelial cadherin) on the cell membrane lateral to the limbal stem cells, act to convert mesenchymal cells into corneal epithelium during embryonic periods, and play an important role in regulating the collective and directional movement of large epithelial sheets during healing of epithelial wounds. Notably, LNCs express E-cadherin more strongly than LSCs, which may explain why LNCs support corneal epithelial proliferation better. It is noted that LNCs may differentiate after prolonged culture in serum-rich media. According to a previous report, an improved embryonic stem cell culture medium can maintain the undifferentiated state of the Niche cells and maintain the expression of embryonic stem cell markers, and in future experiments, LNCs and LSCs need to be cultured and compared under the condition of the improved embryonic stem cell culture medium.

In summary, this study reports a novel method for isolating limbal Niche cells that compares the differences in the phenotype of LNCs and LSCs in supporting limbal stem cell growth, corneal epithelial cell sheet regeneration, and both cellular genes. The results show that limbal Niche cells may become the first choice for treating limbal stem cell deficiencies in the future.

The above examples only show two embodiments of the present invention, and the description thereof is specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (5)

1. A separation culture method of human cornea Niche cells is characterized by comprising the following steps:

tearing off the endothelial layer of the corneal flap left after the plating layer cornea transplantation, and digesting the corneal flap with Dispase solution in water bath at 37 ℃;

then EDTA solution is used for incubation at room temperature to obtain cell suspension;

adding a KCM culture solution into the cell suspension for culturing;

and when the ready-made fibroid cells appear around the corneal epithelial clone, scraping the epithelial cells to selectively retain the fibroid cells, thus obtaining the primary corneal Niche cells.

2. The isolated culture method of human cornea Niche cells of claim 1, wherein the enzyme activity in the Dispase solution is 2.4U/ml.

3. The isolated culture method of human cornea Niche cells of claim 1, wherein the time of the water bath digestion is 1 hour.

4. The method for isolating and culturing human corneal Niche cells according to claim 1, wherein the concentration of the EDTA solution is 0.02%.

5. The method for isolated culture of human cornea Niche cells of claim 1, wherein the incubation time is 2 minutes.

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