Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof

Abstract

Provided is a new type serum-free medium. The medium comprises: DMEM with high glucose (the content of glucose being 4.5 g/L), B27, recombinant human basic fibrolast growth factor (b-FGF), nicotinamide, N-2, vinblastine III (conophylline), non-essential amino acid (NEAA), heparin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), a serum replacement (SR), an insulin-transferrin-selenium complex (ITS), and pentagastrin. Inducing differentiation of mesenchymal stem cells into insulin-secretion-like cells can be achieved in six days in one step using the medium.

Claims

1. A serum-free medium for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-secreting like cells, wherein in 100 parts by volume, the serum-free medium consists of 85-95 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5-8 parts by volume of serum replacement, 1-4 parts by volume of B27 serum-free supplement (50×), 1-1.5 parts by volume of insulin-transferrin-selenium, 0.5-2 parts by volume of aqueous solution of non-essential amino acids, 0.5-2 parts by volume of N-2 serum-free supplement (100×), and heparin in a final concentration of 0.5-2 ug/ml, conophylline in a final concentration of 50-200 ng/ml, nicotinamide in a final concentration of 5-20 mmol/L, and recombinant human basic fibroblast growth factor, epidermal growth factor, hepatocyte growth factor and pentagastrin, each in a final concentration of 5-20 ng/ml; and wherein the aqueous solution of non-essential amino acids comprises glycine, alanine, L-asparagine, L-aspartic acid, glutamic acid, proline and serine, each in a concentration of 8-12 mM, and wherein the serum-free medium induces differentiation of a maximum number of insulin-secreting like cell clusters from the umbilical cord mesenchymal stem cells at 6 days.

2. The serum-free medium according to claim 1, wherein in 100 parts by volume, the serum-free medium consists of 89 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5 parts by volume of serum replacement, 2 parts by volume of B27 serum-free supplement (50×), 1 part by volume of insulin-transferrin-selenium (ITS), 1 part by volume of aqueous solution of non-essential amino acids, 1 part by volume of N-2 serum-free supplement (100×), and heparin in a final concentration of 1 μg/ml, conophylline in a final concentration of 100 ng/ml, nicotinamide in a final concentration of 10 mmol/L, and recombinant human basic fibroblast growth factor, epidermal growth factor, hepatocyte growth factor and pentagastrin each in a final concentration of 10 ng/ml.

3. The serum-free medium according to claim 1, wherein the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells of human origin.

4. A method for preparing the serum-free medium according to claim 1, the method including: mixing medium ingredients until evenly dispersed.

5. A method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-secreting like cells, the method including: culturing the umbilical cord mesenchymal stem cells with the serum-free medium according to claim 1.

6. The method according to claim 5, wherein the umbilical cord mesenchymal stem cells are umbilical cord mesenchymal stem cells of human origin.

7. IThe method according to claim 5, wherein the method includes the following steps: (1) inoculating umbilical cord mesenchymal stem cells in the serum-free medium in a ultra-low attachment six well plate at a density of 5×104 cells/cm.sup.2 and culturing the cells at 37° C., 5% CO.sub.2; (2) every 2 days, centrifuging the cell culture of step (1) at 500 rpm for 4 min, removing the medium, and replacing with a fresh batch of the serum-free medium; (3) culturing for 6-7 days, and harvesting cells; and (4) detecting the cells harvested in step (3) for effect of differentiation induction.

8. The method according to claim 5, wherein the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells isolated from a fresh umbilical cord tissue of a healthy newborn delivered naturally or by cesarean section.

9. The method according to claim 5, wherein the method includes the following steps: (1) inoculating umbilical cord mesenchymal stem cells in the serum-free medium at a density of 2-6×104 cells/cm.sup.2 and culturing the cells at 37° C., 5% CO.sub.2; (2) every 2-3 days, centrifuging the cell culture of step (1) at 300-800 rom for 4 min, removing the medium, and replacing with a fresh batch of the serum-free medium; (3) culturing for 6-7 days, and harvesting cells; and (4) detecting the cells harvested in step (3) for effect of differentiation induction.

10. The method according to claim 9, wherein the step (4) comprises detecting one or more items selected from the group consisting of: amount of insulin released per cell; expression of insulin release-related genes PDX-1, INSULIN and NGN-3; presence of cell nuclear protein PDX-1 and cytosol protein Insulin; dithizone staining; and positive expression rates of specific markers PDX-1, NKX6.1 and insulin of insulin-secreting like cells.

11. The serum-free medium according to claim 1, wherein the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells isolated from a fresh umbilical cord tissue of a healthy newborn delivered naturally or by cesarean section.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings in detail, in which:

(2) FIG. 1 shows a trend chart of the number of cell clusters (>50 μm in diameter) over culture time during screening for the composition of the medium.

(3) FIG. 2 shows a comparison of amounts of insulin released per cell induced in media with different concentrations of pentagastrin.

(4) FIG. 3 shows a comparison of cellular morphology of cells induced in media with different concentrations of ITS, in which panel 3A shows cellular morphology of cells induced for 6 days in medium with 0.5 parts by volume of ITS; panel 3B shows cellular morphology of cells induced for 6 days in medium with 1.0 part by volume of ITS; and panel 3C shows cellular morphology of cells induced for 6 days in medium with 2.0 parts by volume of ITS.

(5) FIG. 4 shows a trend chart of the number of cell clusters (>100 μm in diameter) over culture time during inducing umbilical cord mesenchymal stem cells to differentiate into insulin-secreting like cells using the medium of the present invention (within 10 days).

(6) FIG. 5 shows cellular morphologic changes of cell clusters during inducing umbilical cord mesenchymal stem cells to differentiate into insulin-secreting like cells using the medium of the present invention (within 6 days).

(7) FIG. 6 shows analysis results of specific molecules on the surface of insulin-secreting like cells obtained by inducing with the medium of the present invention by flow cytometry, in which panel 6A shows changes of PDX-1 before and after the induction; panel 6B shows changes of NKx6.1 before and after the induction; panel 6C shows changes of insulin before and after the induction, with panels on the left showing the results before the induction and panels on the right the results after induction for 6 days.

(8) FIG. 7 shows RT-PCR analysis results at transcription level of specific genes of insulin-secreting like cells obtained by inducing umbilical cord mesenchymal stem cells with the medium of the present invention, in which panel 7A shows the results before the induction and panel 7B shows the results after induction for 6 days. In FIG. 7, M represents nucleic acid molecular weight marker, 1 represents internal reference gene GAPDH, 2 represents PDX-1, 3 represents INSULIN, and 4 represents NGN-3.

(9) FIG. 8 shows analysis results at protein level of specific genes of insulin-secreting like cells obtained by inducing umbilical cord mesenchymal stem cells with the medium of the present invention, in which panel 8A shows staining result of nuclear protein PDX-1, panel 8B shows staining result of cytosol protein Insulin, and panel 8C shows a merge of panels 8A and 8B.

(10) FIG. 9 shows the detection of the amounts of insulin released per 10.sup.4 insulin-secreting like cells after stimulating with low glucose and high glucose respectively, in which the insulin-secreting like cells are obtained by inducing umbilical cord mesenchymal stem cells with the medium of the present invention. In FIG. 9, the negative group is cells before induction, the induction group is cells after induction for 6 days, and the positive control group is insulinoma cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(11) The present invention will be further described in detail in combination with the specific embodiments hereinafter. It will be appreciated by those skilled in the art that the embodiments provided are only used to illustrate the present invention, rather than limiting the scope of the present invention in any way.

(12) Experimental methods in the following examples, if no any other special instruction is provided, are all conventional methods. Medicinal materials and agents used in the following examples, if no any other special instruction is provided, are conventional products that can be commercially available.

(13) NEAA used in following examples is an aqueous solution of non-essential amino acids comprising glycine, alanine, L-aspartamide, L-aspartic acid, glutamic acid, proline and serine each in a concentration of 10 mM.

Example 1: Screening for the Composition of the Medium

(14) Basal culture medium: DMEM (with 4.5 g/L glucose)+5% SR+1% NEAA+2% B27 (50×)+1% N-2 (100×)+10 ng/ml HGF+10 ng/ml EGF+10 ng/ml b-FGF+10 mmol/L Nicotinamide.

(15) Ingredients to be screened: 100 ng/ml conophylline, 1 part by volume of ITS, heparin in a final concentration of 1 μg/ml, and betacellulin, Exendi-4, insulin-like growth factor 1 (IGF-1) and pentagastrin each in a final concentration of 10 ng/ml, added to the basal culture medium. Ingredients comprised in each group of medium to be tested were shown in Table 3.

(16) TABLE-US-00003 TABLE 3 Basal culture Conophylline Pentagastrin Heparin ITS betacellulin Exendi-4 IGF-1 medium 100 ng/ml 10 ng/ml 1 μg/ml 1% 10 ng/ml 10 ng/ml 10 ng/ml Group 1 ✓ Group 2 ✓ ✓ Group 3 ✓ ✓ ✓ Group 4 ✓ ✓ ✓ ✓ Group 5 ✓ ✓ ✓ ✓ ✓ Group 6 ✓ ✓ ✓ ✓ ✓ ✓ Group 7 ✓ ✓ ✓ ✓ ✓ ✓ ✓ Group 8 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

(17) In a biosafety cabinet, the third generation hUC-MSCs isolated from Wharton's jelly tissue of umbilical cord of a newborn delivered naturally were inoculated into a ultra-low attachment six well plate at a density of 5×10.sup.4 cells/cm.sup.2, then 2 ml of one of the media to be tested as shown in Table 3 was added in one well and the growth of the cells and the amount of cell clusters formed by the cells were observed.

(18) Results: a small number of cell clusters formed in the medium of group 1; in the media of groups 2, 3 and 4 which had conophylline, pentagastrin and heparin added in respectively, the cell clusters showed an increased number but loose structures; the medium of group 5 had the largest number of cell clusters whose maturity was the best also; while in the media of groups 6, 7 and 9 which had betacellulin, Exendi-4 and insulin-like growth factor 1 added in respectively, little effect on the increase in the number of cell clusters was shown by those three ingredients. The results are shown in FIG. 1.

Example 2: Screening for the Content of Ingredient (Conophylline) in the Medium

(19) Media to be tested: 89 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50×), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), 1 part by volume of N-2 (100×), and heparin in a final concentration of 1 μg/ml, 10 mmol/L nicotinamide, and recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and pentagastrin each in a final concentration of 10 ng/ml, and conophylline with a concentration of 1, 10, 50, 100, 200, and 300 ng/ml respectively.

(20) Human umbilical cord mesenchymal stem cells were induced to differentiate through being cultured in the above media comprising different concentrations of conophylline respectively.

(21) Results: in the two groups of media to be tested which comprise 1 ng/ml and 10 ng/ml conophylline respectively, free cells were found to attach to the edge of cell clusters, and the cell clusters became loose with the prolonging culture time, indicating that the maturity of the cell clusters was insufficient; in the three groups of media to be tested which comprise 50 ng/ml, 100 ng/ml and 200 ng/ml conophylline respectively, cell clusters were compact and grew gradually, indicating that the cell clusters were in a good proliferation; while in the group of medium comprising 300 ng/ml conophylline, the color of the cell clusters was deeper, but the cells at the edge of the cell clusters had changed morphology, indicating that the differentiated cells had undergone an undirected differentiation.

Example 3: Screening for the Content of Ingredient (Pentagastrin) in the Medium

(22) Media to be tested: 89 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50×), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), heparin in a final concentration of 1 μg/ml, 1 part by volume of N-2 (100×), 100 ng/ml conophylline, 10 mmol/L nicotinamide, recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) each in a final concentration of 10 ng/ml, and pentagastrin with a concentration of 1, 2, 5, 10, 20, 30, and 50 ng/ml respectively.

(23) Human umbilical cord mesenchymal stem cells were induced to differentiate through being cultured in the above media comprising different concentrations of pentagastrin respectively, and subsequently the cells obtained from the groups were compared using a glucose stimulation experiment which detects the amount of insulin released per cell (1×10.sup.4 cells in this case). The glucose stimulation experiment was conducted as follows: cell clusters were collected when the cells had been cultured for 6 days, added into a stimulating liquid, 2 ml DMEM with 25 mM/L glucose, and then were blown gently and mixed well; and supernatant was collected after stimulation for 2 hours at 37° C., detected with an insulin ELISA kit, and OD450 value was read finally.

(24) Results: the cells differentiated in the media comprising pentagastrin concentrations of 5, 10, and 20 ng/ml respectively had higher amounts of insulin released per cell. The results are shown in FIG. 2.

Example 4: Screening for the Content of Ingredient (ITS) in the Medium

(25) Media to be tested: 89 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50×), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), heparin in a final concentration of 1 μg/ml, 1 part by volume of N-2 (100×), 100 ng/ml conophylline, 10 mmol/L nicotinamide, and recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and pentagastrin each in a final concentration of 10 ng/ml, and ITS with a concentration of 0.2, 0.5, 0.8, 1.0, 1.2, 1.5, and 2.0 ng/ml respectively.

(26) Human umbilical cord mesenchymal stem cells were induced to differentiate through being cultured in the above media comprising different concentrations of ITS respectively.

(27) Results: in the media comprising 0.2, 0.5, and 0.8 ng/ml ITS respectively, the cells were loose and even died, cell clusters formed were uneven and had smaller diameters, and many dead cells were found at the bottom of the plate; in the media comprising 1.0, 1.2 and 1.5 ng/ml ITS respectively, the cells tightly clustered; while in the medium comprising 2.0 ng/ml ITS, cell clusters formed were compact, but dead cells began to appear and showed an gradually increased number. The results are shown in FIG. 3.

Example 5: Screening for Induction Time

(28) Preparation of the serum-free medium for inducing differentiation:

(29) Composition: 89 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50×), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), 1 part by volume of N-2 (100×), heparin in a final concentration of 1 μg/ml, 100 ng/ml conophylline, 10 mmol/L nicotinamide, and recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and pentagastrin each in a final concentration of 10 ng/ml.

(30) The above ingredients were mixed with each other well to prepare the medium.

(31) Culture of Cells:

(32) In a biosafety cabinet, the third generation hUC-MSCs isolated from Wharton's jelly tissue of umbilical cord of a newborn delivered naturally were inoculated into a ultra-low attachment six well plate at a density of 5×10.sup.4 cells/cm.sup.2, then the plate was transferred to a constant temperature incubator at 37° C., 5% CO.sub.2 after 2 ml of the medium of the present invention was added in each well. Cell suspension in the medium was pipetted into a centrifuge tube slowly, centrifuged at 500 rpm for 4 min at a low speed, then the medium was removed and replaced with fresh serum-free medium of the present invention, with the serum-free medium renewed every 2 days. The amount of cell clusters formed by the cells was observed and counted during continuous induction for 10 days.

(33) Results: the number of cell clusters gradually increased in the first six days since the induction started, reached the maximum (223) on the sixth day, and gradually decreased after the sixth day. The results are shown in FIG. 4.

Example 6: Preparation and Application of the Serum-Free Medium

(34) Preparation of the serum-free medium for inducing differentiation:

(35) Composition: 89 parts by volume of DMEM (High Glucose, with 4.5 g/L glucose), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50×), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), 1 part by volume of N-2 (100×), heparin in a final concentration of 1 μg/ml, 100 ng/ml conophylline, 10 mmol/L nicotinamide, and recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and pentagastrin each in a final concentration of 10 ng/ml.

(36) The above ingredients were mixed with each other well to prepare the medium. The culture of cells was conducted according to the method as described in Example 5, and then cellular morphologic change of cell clusters was observed for 6 days continuously.

(37) Results: after the first day of the induction, the cells agglomerated and formed small cell clusters which were in a large number and had high light transmission; with the culture time prolonged, the small cell clusters gradually converged into large cell clusters, the number of small cell clusters decreased, and the number of large cell clusters gradually increased, and as the diameter of the cell clusters became larger, the light transmission of the cell clusters gradually decreased; and after 6 days of the culture, large cell clusters (mature cell clusters) occupied most of the field of view. The results are shown in FIG. 5.

Example 7: Analysis of Specific Markers on the Surface of Insulin-Secreting Like Cells by Flow Cytometry

(38) After differentiation induction for 6 days, cell clusters in Example 6 were collected in a centrifuge tube, and then centrifuged at 500 rpm for 4 minutes at 4° C. The supernatant was discarded, the cell clusters remained were collected, and 0.25% trypsin was added in while gently pipetting and mixing for 20 min. The digestion was stopped by adding fresh medium when the structure of the cell clusters was loose and no suspended cell cluster could be seen by naked eyes, and then the tube was centrifuged at 1200 rpm for 6 minutes. The supernatant was discarded and the cells were collected and washed twice with PBS. The cells were then transferred to tubes for flow cytometry in an amount of 1×10.sup.5 cells per tube, and 500 μL of 4% paraformaldehyde was added into each tube for immobilization for 30 min at room temperature, and then the tubes were centrifuged at 1200 rpm for 6 minutes. The supernatant was discarded, the cells were collected and 5 μL of each of antibodies against PDX-1, NKX6.1, Insulin, and IgG1-PE (isotype control) was added into one of the tubes respectively. The cells were mixed well and incubated in dark for 30 minutes at 4° C., washed once with PBS, centrifuged and the supernatant was discarded. The collected cells were resuspended by addition of 500 μL PBS, and then detected on a Flow Cytometer (Flow Cytometer XL, Beckman). 1×10.sup.4 cells were collected from each sample.

(39) Results: the mesenchymal stem cells expressed specific markers of insulin-secreting like cells after being induced for 6 days, in which the positive expression rate of PDX-1 detected by Flow Cytometer was 51.4%, the positive expression rate of NKx6.1 was 15.36%, and the positive expression rate of insulin was 24.03%. The cells before induction did not express those specific markers. The results are shown in FIG. 6.

Example 8: RT-PCR Analysis of Insulin-Secreting Like Cells Specific Genes

(40) After differentiation induction for 6 days, cell clusters in Example 6 were collected in a centrifuge tube, and then RNAs were extracted according to the instruction of Total RNA Kit I (R6834-01, OMRGA). The extracted RNAs were reversely transcribed to obtain cDNA samples using a reverse transcription kit (RR014A, TAKARA), and then the cDNA samples were subjected to PCR amplification. After agarose gel electrophoresis, a gel imager was used for observation.

(41) Results: the cells expressed insulin-secreting like cells specific genes after being induced, and bands of PDX-1, INSULIN and NGN-3 with varying brightness could be seen. The results are shown in FIG. 7.

Example 9: Analysis of hUC-MSCs Specific Proteins by Immunofluorescence Staining

(42) After differentiation induction for 6 days, cell clusters in Example 6 were collected in a centrifuge tube, immobilized for 15 min with 4% paraformaldehyde, and then treated with 0.25% TritonX-100 for 20 min. Diluted red mouse anti-human antibody (anti-Insulin antibody) was added in after the cells were blocked with goat serum, and then incubated with the cells in dark overnight at 4° C. Afterwards, nuclear protein PDX-1 was stained, the cells were incubated in dark overnight at 4° C., and then observed under a fluorescence microscope.

(43) Results: insulin-secreting like cells were obtained by inducing with the method of the present invention which expressed INSULIN and PDX-1 specific proteins. The results are shown in FIG. 8.

Example 10: Measurement of Amount of Insulin Released Per Cell

(44) After differentiation induction for 6 days, cell clusters in Example 6 were collected in a centrifuge tube, centrifuged and the supernatant was discarded. The cell clusters were resuspended in PBS, aspirated gently, and then centrifuged at 500 rpm for 3 minutes; the PBS was discarded and a stimulating liquid, DMEM with 5.5 mM/L glucose (group of low glucose) or DMEM with 25 mM/L glucose (group of high glucose) was added into the tube respectively. Afterwards, the cell clusters were blown gently and mixed well, and stimulated for 2 hours at 37° C., 5% CO.sub.2. The tube was centrifuged at 500 rpm for 3 minutes, and the supernatant was collected for detection. The amount of insulin released was measured by ELISA, and a group of insulinoma cells was served as a positive control group.

(45) Results: insulin-secreting like cells were obtained by inducing with the method of the present invention which released 4.393 ulU/ml insulin per 10.sup.4 cells under stimulation of high glucose, and the cells of the positive control group released 6.9828 ulU/ml insulin per 10.sup.4 cells under stimulation of high glucose. That is, the amount of insulin released by the cells of induction group equals to 62.91% of the positive group. The results are shown in FIG. 9.

(46) The above description for the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and variations according to the present invention, which are within the protection scope of the present invention without departing from the spirit of the same.