Serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell

Abstract

A serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell in the field of differentiation induction of stem cells, prepared by the following method: uniformly mixing the serum-free complete medium, containing 5-10 ?mol of resveratrol, 2-4 ?mol of icariin, 1-3 nmol of aspirin, 1-3 nmol of parathyroid hormone, 5-10 nmol of hydrocortisone, 1-3 mg of rapamycin, 2-10 ?g of testosterone, 2-10 ?g of EPO, 2-10 ?g of LIF and the balance of a corneal epithelial cell serum-free medium in per 1 L; and then performing sterilization by filtration. The disclosure uses resveratrol and icariin in combination with aspirin, parathyroid hormone, hydrocortisone, rapamycin, testosterone and growth factors to cooperatively induce directional differentiation, uses nontoxic induction components, is high in induction efficiency and short in induction time, and achieves high induced corneal epithelial cell activity, no cell transplantation rejection, no ethical problem and high safety.

Claims

1. A serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell, prepared by the following method: uniformly mixing the serum-free complete medium for differentiation induction, containing 5-10 ?mol of resveratrol, 2-4 ?mol of icariin, 1-3 nmol of acetylsalicylic acid, 1-3 nmol of parathyroid hormone, 5-10 nmol of hydrocortisone, 1-3 mg of rapamycin, 2-10 ?g of testosterone, 2-10 ?g of erythropoietin (EPO), 2-10 ?g of leukemia inhibitory factor (LIF) and the balance of a corneal epithelial cell serum-free medium in per 1 L; and then performing sterilization by filtration.

2. A serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell, prepared by the following method: uniformly mixing the serum-free complete medium for differentiation induction, containing 8 ?mol of resveratrol, 3 ?mol of icariin, 2 nmol of acetylsalicylic acid, 2 nmol of parathyroid hormone, 7 nmol of hydrocortisone, 2 mg of rapamycin, 7 ?g of testosterone, 7 ?g of erythropoietin (EPO), 7 ?g of leukemia inhibitory factor (LIF) and the balance of a corneal epithelial cell serum-free medium in per 1 L; and then performing sterilization by filtration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The sole FIGURE shows morphology (?400) of corneal epithelial cells appeared after differentiation of mesenchymal stem cells induced by a medium of the disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(2) Experiment methods in the following examples are conventional methods unless otherwise specified. Instruments and reagents for the experiments are commercially available.

(3) Example 1 All components of a serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell of the disclosure were commercially available: resveratrol, trademark: Sigma, and product code: R5010; icariin, trademark: Shanghai Weijing Biology, and product code: 489-32-7; aspirin, trademark: Sigma, and product code: A2093-100G; parathyroid hormone, trademark: Sigma, and product code: P7036; hydrocortisone, trademark: Sigma, and product code: H3160; rapamycin, trademark: TargetMol, and product code: T1537; testosterone, trademark: Sigma, and product code: T1500; EPO (hemopoietin), trademark: PeproTech, and product code: CYT-201; LIF (leukaemia inhibitory factor), trademark: PeproTech, and product code: 96-300-05-5; and corneal epithelial cell serum-free medium (CEpiCM), trademark: ScienCell, and product code: 6511.

(4) A serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell was prepared from the following components respectively according to the following concentration proportion: per 1 L of the serum-free complete medium for differentiation induction contained 8 ?mol of resveratrol, 3 ?mol of icariin, 2 nmol of aspirin, 2 nmol of parathyroid hormone, 7 nmol of hydrocortisone, 2 mg of rapamycin, 7 ?g of testosterone, 7 ?g of EPO, 7 ?g of LIF and the balance of a corneal epithelial cell serum-free medium. After being uniformly mixed, the components were sterilized by filtration.

(5) Example 2 A serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell was prepared from the following components respectively according to the following concentration proportion: per 1 L of the serum-free complete medium for differentiation induction contained 5 ?mol of resveratrol, 2 ?mol of icariin, 1 nmol of aspirin, 1 nmol of parathyroid hormone, 5 nmol of hydrocortisone, 1 mg of rapamycin, 2 ?g of testosterone, 2 ?g of EPO, 2 ?g of LIF and the balance of a corneal epithelial cell serum-free medium. After being uniformly mixed, the components were sterilized by filtration.

(6) Example 3 A serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell was prepared from the following components respectively according to the following concentration proportion: per 1 L of the serum-free complete medium for differentiation induction contained 10 ?mol of resveratrol, 4 ?mol of icariin, 3 nmol of aspirin, 3 nmol of parathyroid hormone, 10 nmol of hydrocortisone, 3 mg of rapamycin, 10 ?g of testosterone, 10 ?g of EPO, 10 ?g of LIF and the balance of a corneal epithelial cell serum-free medium. After being uniformly mixed, the components were sterilized by filtration.

(7) Example 4 By taking human adipose-derived mesenchymal stem cells as an example, the induction medium prepared in Example 1 was used for performing a corneal epithelial cell differentiation induction experiment on the mesenchymal stem cells. The steps were as follows:

(8) Adipose-derived mesenchymal stem cells were induced to differentiate into corneal epithelial cells: 3.sup.rd-generation MSC was inoculated into a 6-hole cell culture plate previously loaded with polylysine-treated sterile cover glasses to prepare round coverslips. Differentiation induction was performed when the cells were fused to about 80% and grew vigorously. Experiment groups are shown in table 1:

(9) TABLE-US-00001 TABLE 1 Experiment groups Groups Induction conditions Induction Induction for 5 d by the induction medium of the group of the disclosure. disclosure Conditioned Preparation of a conditioned medium: human limbal stem medium cells were cultured by a DMEM/F12 medium containing group 20% FBS + 10 ?g/L EGF in a 37? C. 5% CO.sub.2 incubator. The medium was changed once every 2 d. The medium changed in each time was collected and transferred into a centrifuge tube to be centrifugated at 1000 rpm/min for 5 min. Supernatants were collected in a sterile tube to be put into a ?20? C. refrigerator for use. Induction was performed for 30 d by using the above conditioned medium. Co-culture According to instructions of Millipore Company, a group Transwell co-culture system was used. The human limbal stem cells were inoculated into Transwell chambers and were then put into the 6-hole cell culture plate for co- culture and induction for 7 d together with the mesenchymal stem cell.

(10) After induction, the cells were identified. CK3, CK12 and CK19 were selected to be used as corneal epithelial cell markers. Expression conditions of CK3, CK12 and CK19 before and after the induction were detected by an immunocytochemical method. 4 cover glasses were randomly extracted, 5 sights were randomly selected in a 200-time field, and the proportion of positive cells was respectively calculated. The proportion of the positive cells in total cells was expressed by mean?standard deviation (x?s). Statistical analysis was performed by SPSS11.0 software. Experiment data was expressed by (x?s). Variance analysis was adopted for comparison among a plurality of groups. x.sup.2 inspection was adopted for rate comparison between two groups. P<0.05 showed existence of statistical significance. Induction results of the three groups are as shown in Table 2:

(11) TABLE-US-00002 TABLE 2 Induction results of three groups (n = 20, x ? s) Immunocytochemical method Groups CK3 CK12 CK19 Induction group of the (92.4 ? 1.3)% (89.5 ? 1.6)% (93.7 ? 1.1)% disclosure Conditioned culture (32.5 ? 0.9)% (29.6 ? 0.8)% (24.4 ? 0.5)% group Co-culture group (41.6 ? 1.5)% (37.5 ? 2.0)% (44.2 ? 1.7)%

(12) As shown in Table 2, the positive cell ratio of CK3, CK12 and CK19 after the differentiation induction of the induction group of the disclosure was higher than that of the conditioned medium group and that of the co-culture group (P<0.05). It showed that the induction efficiency of the medium of the disclosure was obviously higher than that of the conditioned culture group and that of the co-culture group, and reached 90% or higher.

(13) Based on the above, the serum-free complete medium for inducing differentiation of a mesenchymal stem cell to a corneal epithelial cell of the disclosure has a highest induction efficiency and shortest induction time, and is worthy of popularization.