SERUM-FREE POLYPEPTIDE COMPOSITION FOR PROMOTING PROLIFERATION OF MESENCHYMAL STEM CELLS

Abstract

The present invention provides a serum-free polypeptide composition for promoting proliferation of mesenchymal stem cells. The composition mainly comprises: 10-100 μg/L of tripeptide-1; 1-20 μg/L of tripeptide-2, 1-20 μg/L of hexapeptide-9, 1-20 μg/L of palmitoyl hexapeptide-12; and 10-100 μg/L of a laminin-derived peptide. The serum-free polypeptide composition provided in the present disclosure has clear chemical components without animal origins or serum, can achieve rapid proliferation of the mesenchymal stem cells, and maintains the biological characteristics and immunophenotypic stability of the mesenchymal stem cells while solving the problem of the insufficient quantity of cells.

Claims

1. A serum-free polypeptide composition for promoting proliferation of mesenchymal stem cells, comprising: TABLE-US-00012 tripeptide-1 1~20 μg/L; tripeptide-2 1~20 μg/L; hexapeptide-9 1~20 μg/L; palmitoyl hexapeptide-12 1~20 μg/L; laminin-derived peptide 10~100 μg/L; non-essential amino acids 1~5 vol %; glutamine 1~4 mmol/L; lipid mixture 1~5 vol %; ITS (Insulin, Transferrin, Selenium) 1~5 vol %; recombinant human serum albumin 1~5 g/L; recombinant human epidermal growth factor 5~25 μg/L; recombinant human fibronectin 5~15 μg/L; L-glutathione 1~5 mg/L; β-mercaptoethanol 1~5 mg/L; and α-MEM basal medium 10.2 g/L.

2. The serum-free polypeptide composition according to claim 1, wherein content of the tripeptide-1 is 3-18 μg/L.

3. The serum-free polypeptide composition of claim 1, wherein content of the tripeptide-2 is 3-18 μg/L.

4. The serum-free polypeptide composition of claim 1, wherein content of the hexapeptide-9 is 4-18 μg/L.

5. The serum-free polypeptide composition of claim 1, wherein content of the palmitoyl hexapeptide-12 is 3-17 μg/L.

6. The serum-free polypeptide composition of claim 1, wherein content of the laminin-derived peptide is 30-65 μg/L.

7. The serum-free polypeptide composition of claim 1, wherein content of the tripeptide-1 is 10 μg/L, content of the tripeptide-2 is 10 μg/L, content of the hexapeptide-9 is 10 μg/L, content of the palmitoyl hexapeptide is 10 μg/L, and content of the laminin-derived peptide is 60 μg/L.

8. The serum-free polypeptide composition according to claim 7, wherein content of the non-essential amino acid is 1 vol %, content of the glutamine is 1 to 4 mmol/L, content of the lipid mixture is 1 vol %, content of the ITS is 1 vol %, content of the recombinant human serum albumin is 2.5 g/L, content of the recombinant human epidermal growth factor is 20 μg/L, content of the recombinant human fibronectin is 10 μg/L, content of the L-glutathione is 2 mg/L, and content of the β-mercaptoethanol is 2 mg/L.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 is a morphological images (40×) of UC-MSCs in the experiment group and the control group of the present disclosure;

[0020] FIG. 2 is a graph of the growth curve of UC-MSCs in the experiment groups and the control groups of the present disclosure;

[0021] FIG. 3 is a diagram of the adipogenic differentiation results of UC-MSCs in the experiment groups and the control groups of the present disclosure (400×); and

[0022] FIG. 4 is a diagram of the osteogenic differentiation results of UC-MSCs in the experiment groups and the control groups of the present disclosure (40×).

DETAILED DESCRIPTION

[0023] In order to make the technical solutions of the present disclosure more clearly understood, the preferred embodiments of the present disclosure will be described below in conjunction with examples. It should be understood that these descriptions are only for further illustrating the features and advantages of the present disclosure, rather than limiting the claims of the present disclosure.

[0024] Aiming at the serum-free medium required for mesenchymal stem cells in the prior art, this application provides a polypeptide composition, which can promote the rapid proliferation of mesenchymal stem cells as a serum-free medium. Specifically, an embodiment of the present disclosure discloses a serum-free polypeptide composition for promoting the proliferation of mesenchymal stem cells, comprising:

TABLE-US-00002 tripeptide-1 1~20 μg/L; tripeptide-2 1~20 μg/L; hexapeptide-9 1~20 μg/L; palmitoyl hexapeptide-12 1~20 μg/L; laminin-derived peptide 10~100 μg/L; non-essential amino acids 1~5 vol %; glutamine 1~4 mmol/L; lipid mixture 1~5 vol %; ITS 1~5 vol %; recombinant human serum albumin 1~5 g/L; recombinant human epidermal growth factor 5~25 μg/L; recombinant human fibronectin 5~15 μg/L; L-glutathione 1~5 mg/L; β-mercaptoethanol 1~5 mg/L; α-MEM basal medium 10.2 g/L.

[0025] In the above medium, tripeptide-1, tripeptide-2, hexapeptide-9, palmitoyl hexapeptide-12 and laminin-derived peptides are used as a polypeptide combination of the polypeptide composition. Among them, tripeptide-1 can promote the production of elastin and collagen. The α-MEM basal medium is used as a constant volume substrate, and content of tripeptide-1 is 1-20 μg/L; in a specific embodiment, content of the tripeptide-1 is 3-18 μg/L.

[0026] The tripeptide-2 is an active tripeptide derived from an elastase inhibitor, which can reduce the synthesis of progerin, and its content is 1-20 μg/L. In a specific embodiment, content of the tripeptide-2 is 3˜18 μg/L.

[0027] The hexapeptide-9 is synthesized from six amino acids and is a very stable collagen peptide, which can promote the production of cellular collagen, laminin and integrin; its content is 1-20 μg/L. In a specific embodiment, content of the hexapeptide-9 is 4-18 μg/L.

[0028] The palmitoyl hexapeptide-12 has a chemotactic effect and can promote the migration and proliferation of dermal fibroblasts and synthesis of matrix macromolecules; its content is 1-20 μg/L. In a specific embodiment, content of the palmitoyl hexapeptide-12 is 3˜17 μg/L.

[0029] The laminin-derived peptide has functions similar to laminin and can promote cell adhesion and proliferation; its content is 10-100 μg/L. In a specific embodiment, content of the laminin-derived peptide is 30˜65 μg/L.

[0030] The above-mentioned tripeptide-1, tripeptide-2, hexapeptide-9, palmitoyl hexapeptide-12 and laminin-derived peptides gradually increase in performance as the content increases, but their performances are not greatly improved when the contents are beyond the upper limit of the application.

[0031] In the present disclosure, by introducing the above-mentioned polypeptides into the polypeptide composition, the polypeptides synergistically cooperates with the basal medium, so that the polypeptide composition promotes the rapid proliferation of mesenchymal stem cells.

[0032] As a serum-free medium for mesenchymal stem cells culture, the polypeptide composition also comprises a basic serum-free medium, which specifically includes: non-essential amino acids 1˜5 vol %; glutamine 1˜4 mmol/L; lipid mixture 1˜5 vol %; ITS (Insulin, Transferrin, Selenium) 1˜5 vol %; recombinant human serum albumin 1˜5 g/L; recombinant human epidermal growth factor 5˜25 μg/L; recombinant human fibronectin 5˜15 μg/L; L-glutathione peptide 1˜5 mg/L; β-mercaptoethanol 1˜5 mg/L; and α-MEM basal medium 10.2 g/L. The above-mentioned basic serum-free medium is used for the cultivation of mesenchymal stem cells.

[0033] In a specific embodiment, the polypeptide composition includes: the content of the tripeptide-1 is 10 μg/L, the content of the tripeptide-2 is 10 μg/L, the content of the hexapeptide-9 is 10 μg/L, the content of the palmitoyl hexapeptide is 10 μg/L, the content of the laminin-derived peptide is 60 μg/L, the content of the non-essential amino acid is 1 vol %, the content of the glutamine is 1˜4 mmol/L, the content of the lipid mixture is 1 vol %, the content of the ITS is 1 vol %, the content of the recombinant human serum albumin is 2.5 g/L, the content of the recombinant human epidermal growth factor is 20 μg/L, the content of the recombinant human fibronectin is 10 μg/L, the content of the L-glutathione is 2 mg/L, and the content of the β-mercaptoethanol is 2 mg/L.

[0034] The polypeptide composition described in the present disclosure can be prepared according to a method well known to those skilled in the art. After the components are mixed, the mixture is filtered and sterilized by passing the filter membrane, and then added to the α-MEM basal medium and mixed well. After that, the serum-free medium for promoting the proliferation of umbilical cord derived mesenchymal stem cells (UC-MSCs) was obtained.

[0035] The mesenchymal stem cell-promoting polypeptide composition provided by the present disclosure has definite chemical components, no animal origin and no serum. It realizes the rapid proliferation of UC-MSCs, solves the problem of insufficient cell numbers while maintaining the biological characteristics and immunity phenotypic stability of mesenchymal stem cells.

[0036] In order to further understand the present disclosure, the polypeptide composition for promoting the proliferation of mesenchymal stem cells provided by the present disclosure will be described in detail in conjunction with examples below. The protection scope of the present disclosure is not limited by the following examples.

[0037] The components and reagents used in the following examples are all commercial products. For example, α-MEM basal medium (Cat. No. 41060037) and non-essential amino acid solution (Cat. No. 11140-050) were purchased from Gibco; other components can be purchased from Sigma, MP, Gibco or other companies.

EXAMPLE 1

Serum-Free Medium Preparation

[0038] 1) Based on α-MEM basal medium, a serum-free medium was prepared according to the following formula:

TABLE-US-00003 non-essential amino acids 1 vol % glutamine 1~4 mmol/L lipid mixture 1 vol % ITS 1 vol % recombinant human serum albumin 2.5 g/L recombinant human epidermal growth factor 20 μg/L recombinant human fibronectin: 10 μg/L L-glutathione 2 mg/L β-mercaptoethanol 2 mg/L tripeptide-1 10 μg/L tripeptide-2 10 μg/L hexapeptide-9 10 μg/L palmitoyl hexapeptide-12 10 μg/L laminin-derived peptide 50 μg/L α-MEM basal medium 10.2 g/L

[0039] The above-mentioned components were dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present disclosure, namely experiment group 2. The composition was filtered and sterilized through a 0.22 μm filter membrane to promote the proliferation of mesenchymal stem cells.

[0040] 2) Based on α-MEM basal medium, a serum-free medium was prepared according to the following formula:

TABLE-US-00004 non-essential amino acids 0.01 vol % glutamine 1~4 mmol/L lipid mixture 1 vol % ITS 1 vol % recombinant human serum albumin 2.5 g/L recombinant human epidermal growth factor 20 μg/L recombinant human fibronectin: 10 μg/L L-glutathione 2 mg/L β-mercaptoethanol 2 mg/L tripeptide-1 20 μg/L tripeptide-2 20 μg/L hexapeptide-9 20 μg/L palmitoyl hexapeptide-12 20 μg/L laminin-derived peptide 100 μg/L α-MEM basal medium 10.2 g/L

[0041] The above-mentioned components were dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present disclosure, namely experiment group 3. The composition was filtered through a 0.22 μm filter for sterilization and then used for promoting the proliferation of mesenchymal stem cells.

[0042] 3) Based on α-MEM basal medium, prepare basic serum-free medium according to the following formula:

TABLE-US-00005 non-essential amino acids 1 vol % glutamine 1~4 mmol/L lipid mixture 1 vol % ITS 1 vol % recombinant human serum albumin 2.5 g/L recombinant human epidermal growth factor 20 μg/L recombinant human fibronectin: 10 μg/L L-glutathione 2 mg/L β-mercaptoethanol 2 mg/L α-MEM basal medium 10.2 g/L

[0043] The above-mentioned components were dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present disclosure, namely experiment group 1. The composition was filtered and sterilized through a 0.22 μm filter membrane, and then used to promote the proliferation of mesenchymal stem cells.

[0044] 4) Based on α-MEM basal medium, a serum-free medium was according to the following formula:

TABLE-US-00006 non-essential amino acids 1 vol % glutamine 1~4 mmol/L lipid mixture 1 vol % ITS 1 vol % recombinant human serum albumin 2.5 g/L recombinant human epidermal growth factor 20 μg/L recombinant human fibronectin: 10 μg/L L-glutathione 2 mg/L β-mercaptoethanol 2 mg/L tripeptide-1 10 μg/L tripeptide-2 10 μg/L α-MEM basal medium 10.2 g/L

[0045] The above components were dissolved in water at room temperature and fully dissolved to obtain the polypeptide composition of the present disclosure, namely the control group 3. The composition was filtered and sterilized through a 0.22 μm filter membrane, and then used to promote the proliferation of mesenchymal stem cells.

[0046] 5) Based on α-MEM basal medium, a serum-free medium was prepared according to the following formula:

TABLE-US-00007 non-essential amino acids 1 vol % glutamine 1~4 mmol/L lipid mixture 1 vol % ITS 1 vol % recombinant human serum albumin 2.5 g/L recombinant human epidermal growth factor 20 μg/L recombinant human fibronectin: 10 μg/L L-glutathione 2 mg/L β-mercaptoethanol 2 mg/L hexapeptide-9 10 μg/L palmitoyl hexapeptide-12 10 μg/L laminin-derived peptide 50 μg/L α-MEM basal medium 10.2 g/L

[0047] The above-mentioned components were dissolved in water and fully dissolved at room temperature to obtain the polypeptide composition of the present disclosure, namely the control group 4. The composition was filtered through a 0.22 μm filter membrane to be sterilized, and then used for promoting the proliferation of mesenchymal stem cells.

[0048] Experiment group 1, experiment group 2, experiment group 3, control group 3, and control group 4 were set using the medium prepared above, respectively. α-MEM complete medium containing 10% FBS was set as control group 1, STEMPRO® hMSC SFM (GIBCO) was set as the control group 2. The following experiments were performed.

EXAMPLE 2

Morphology Observation and Activity Detection of UC-MSCs

[0049] The P3 generation of UC-MSCs were selected to carry out the experiment. UC-MSCs were inoculated into T25 culture flasks at a density of 1×10.sup.4/cm.sup.2, with 3 replicates in each group. The cells were cultured in a 5% CO.sub.2 incubator at 37° C. for 48 hours and the images of the UC-MSCs were collected, the results were shown in FIG. 1. FIG. 1A showed the morphology of UC-MSCs in control group 1, FIG. 1B showed the morphology of UC-MSCs in control group 2, FIG. 1C showed the morphology of UC-MSCs in experiment group 1, FIG. 1D showed the morphological of UC-MSCs in experiment group 2, and FIG. 1E showed the morphology of UC-MSCs in experiment group 3.

[0050] It can be seen from FIG. 1 that the UC-MSCs of each group grew in a single layer, and most of the cells were long spindle-shaped with irregular morphology. The morphology of

[0051] UC-MSCs in the three experiment groups was more similar to that of the control group 2.

[0052] After cultured for 72 hours, the UC-MSCs of each group were digested with 0.25% trypsin solution, and a cell counter (Countstar) was used to count the number of cells and cell viability in each group was calculated. The results are shown in Table 1.

TABLE-US-00008 TABLE 1 Detection results of UC-MSCs in each group Experiment Group Cell Viability (%) Control Group 1 91.75 ± 0.11 Control Group 2 93.20 ± 0.25 Control Group 3 90.05 ± 0.34 Control Group 4 91.86 ± 0.12 Experiment Group 1 91.25 ± 0.35 Experiment Group 2 98.66 ± 0.21 Experiment Group 3 97.44 ± 0.14

[0053] The cell viabilities of experiment group 2 and experiment group 3 were compared with the control group 1, the control group 3, and the control group 4 respectively, and there were significant differences (p<0.05).

[0054] It can be seen from Table 1 that the UC-MSCs viability of the experiment group is higher than that of the control group 1 and the control group 2, and the UC-MSCs viability of the experiment group 2 and the experiment group 3 is higher than that of the experiment group 1.

EXAMPLE 3

Detection of UC-MSCs Proliferation Rate

[0055] The P3 generation of UC-MSCs were selected to carry out the experiment. UC-MSCs were inoculated in a 24-well plate at a density of 1×10.sup.4 cells/ml and cultured in a 5% CO.sub.2 incubator at 37° C. Cells were collected daily for cell count, and each time 3 wells were collected randomly and calculated, for 7 consecutive days. The cell growth curve was plotted and the results were shown in Table 2 and FIG. 2.

TABLE-US-00009 TABLE 2 7-day cell counting results of UC-MSCs in each group Counts (×10{circumflex over ( )}4) Experiment Group 0 d 1 d 2 d 3 d 4 d 5 d 6 d 7 d Control Group 1 1.00 ± 0.00 1.15 ± 0.09 2.30 ± 0.46 5.00 ± 0.38 9.45 ± 0.40 15.60 ± 1.08 29.25 ± 4.48 31.67 ± 2.23 Control Group 2 1.00 ± 0.00 0.95 ± 0.23 3.00 ± 0.26 5.40 ± 0.44 9.40 ± 0.46 17.25 ± 0.40 32.80 ± 0.26 34.83 ± 1.99 Control Group 3 1.00 ± 0.00 1.07 ± 0.05 2.44 ± 0.23 5.06 ± 0.19 13.28 ± 1.75  26.34 ± 2.61 30.16 ± 1.14 33.35 ± 2.09 Control Group 4 1.00 ± 0.00 1.08 ± 0.02 3.06 ± 0.26 5.02 ± 0.28 13.62 ± 1.30  24.38 ± 1.80 32.85 ± 3.26 35.55 ± 2.70 Experiment Group 1 1.00 ± 0.00 1.00 ± 0.17 1.80 ± 0.53 5.53 ± 0.56 9.70 ± 0.38 16.60 ± 2.42 30.05 ± 1.15 33.20 ± 0.71 Experiment Group 2 1.00 ± 0.00 1.00 ± 0.09 2.70 ± 0.30 7.50 ± 0.40 13.55 ± 0.88  26.88 ± 0.75 40.05 ± 1.05 43.02 ± 1.05 Experiment Group 3 1.00 ± 0.00 1.08 ± 0.11 3.50 ± 0.89 6.52 ± 1.19 12.62 ± 1.84  22.92 ± 1.86 36.09 ± 2.65 40.54 ± 2.90

[0056] Cell mass on day 7 of each group was analyzed for significance. The experiment group 2 and the experiment group 3 were compared with the control group 1, the control group 3 and the control group 4, respectively, and there were significant differences (p<0.05).

[0057] From the results in Table 2 and FIG. 2, compared with the experiment group 1 and the control groups, the UC-MSCs cultured by the polypeptide composition of the present disclosure have higher proliferation activity.

[0058] According to the doubling time calculation formula: DT=t*[lg2/(lgNt−lgNo)], wherein t is culture time; No is the number of cells recorded for the first time; Nt is the number of cells after t passages. The results are shown in Table 3.

TABLE-US-00010 TABLE 3 Doubling time of UC-MSCs in each group Experiment Control Control Control Control Experiment Experiment Experiment Group Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Doubling 32.12 ± 0.15 31.01 ± 0.21 32.22 ± 0.30 32.23 ± 0.32 29.18 ± 0.24 24.77 ± 0.17 26.62 ± 0.33 Time (H)

[0059] The experiment group 2 and the experiment group 3 were compared with the control group 1, the control group 3, and the control group 4 respectively, and there were significant differences (p<0.05).

[0060] It can be seen from Table 3 that the doubling time of UC-MSCs in experiment group 2 and experiment group 3 is less than that in experiment group 1 and the four control groups, indicating that the proliferation rate of UC-MSCs cultured in the polypeptide composition of the present disclosure is higher.

EXAMPLE 4

UC-MSCs Surface Marker Detection

[0061] The P3 generation of UC-MSCs were selected to carry out the experiment. UC-MSCs were inoculated into T25 culture flasks at a density of 1×10.sup.4/cm.sup.2 and cultured in a 5% CO.sub.2 incubator at 37° C. After 3 days, the UC-MSCs of each group were collected by digestion with 0.25% trypsin solution, and the expression of surface markers such as CD105, CD73, CD90, CD34, CD45, HLA-DR, etc. were detected by flow cytometry. The results are shown in Table 4.

TABLE-US-00011 TABLE 4 Surface marker detection results of UC-MSCs in each group Experiment Group CD105 CD90 CD73 CD34 CD45 HLA-DR Control Group 1 100.0% 98.50% 96.35% 0.02% 0.15% 0.01% Control Group 2 99.96% 100.0% 95.24% 0.01% 0.00% 0.03% Control Group 3 100.0% 98.35% 96.76% 0.00% 0.00% 0.02% Control Group 4 97.95% 99.25% 96.33% 0.04% 0.00% 0.02% Experiment Group 1 99.45% 100.0% 98.10% 0.00% 0.01% 0.02% Experiment Group 2 100.0% 97.46% 99.80% 0.01% 0.00% 0.01% Experiment Group 3 100.0% 99.25% 100.0% 0.02% 0.00% 0.01%

[0062] It can be seen from Table 4 that the surface markers CD105, CD73, and CD90 of UC-MSCs are expressed in each experiment groups and control groups, while CD34, CD45, and HLA-DR are not expressed, and there is no significant difference between the groups, indicating that the polypeptide composition of present disclosure does not affect the expression of UC-MSCs surface markers during culturing.

EXAMPLE 5

Detection of Multi-Directional Differentiation Potential of UC-MSCs

[0063] The P3 generation UC-MSCs were selected for the experiment. The UC-MSCs of the experiment groups 1˜3, the control group 1 and the control group 2 were routinely cultured and passaged to the P5 generation, and then inoculated in a 6-well plate at a density of 1×10.sup.5/ml. The cells were incubated at 37° C. in a 5% CO.sub.2 incubator. When the confluence of UC-MSCs in each group reached more than 80%, control wells and induction wells were set respectively and osteogenic and adipogenic differentiation of UC-MSCs were induced. After 14 days, the cells in the adipogenic differentiation experiment group were stained with Oil Red O. And after 21 days, the cells in the osteogenic differentiation experiment group were stained with Alizarin Red. The results are shown in FIG. 3 and FIG. 4.

[0064] FIG. 3A is the adipogenic differentiation result of UC-MSCs of control group 1, FIG. 3B is the adipogenic differentiation result of UC-MSCs of control group 2, and FIG. 3C is the adipogenic differentiation result of UC-MSCs of experiment group 1, FIG. 3D is the adipogenic differentiation result of UC-MSCs of experiment group 2, FIG. 3E is the adipogenic differentiation result of UC-MSCs of experiment group 3.

[0065] FIG. 4A is an image of the osteogenic differentiation result of UC-MSCs in control group 1, FIG. 4B is an image of the osteogenic differentiation result of UC-MSCs in control group 2, FIG. 4C is an image of the osteogenic differentiation of UC-MSCs in experiment group 1, FIG. 4D is an image of the osteogenic differentiation result of UC-MSCs in experiment group 2, and FIG. 4E is an image of the osteogenic differentiation result of UC-MSCs in experiment group 3. The experiment results show that the culture of UC-MSCs supplemented with the polypeptide combination of the present disclosure will not affect their adipogenic and osteogenic differentiation potential and maintain their stemness.

[0066] The description of the above embodiments is only used to help the understanding of the method and the core idea of the present disclosure. It should be pointed out that for those of ordinary skilled in the art, several improvements and modifications can be made to the present disclosure without departing from the principle of the present disclosure, and these improvements and modifications also fall within the protection scope of the claims of the present disclosure.

[0067] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or employ the present disclosure. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments shown in this text, but would conform to the widest scope consistent with the principles and novel features disclosed herein.