Induction liquid and induction method for inducing conversion of multipotent stem cells into mesenchymal stem cells from domestic dogs

Abstract

The invention provides an induction liquid and an induction method for inducing conversion of multipotent stem cells into mesenchymal stem cells from domestic dogs, and belongs to the technical field of cell engineering. The invention provides an induction liquid for inducing conversion of pluripotent stem cells into mesenchymal stem cells from domestic dogs, and the induction liquid takes a DMEM / L culture medium as a basic culture medium, and also comprises 5-15% of FBS. The invention also constructs a method for inducing the induced pluripotent stem cells of the domestic dogs into the mesenchymal stem cells by using the induction liquid, the induced pluripotent stem cells of the domestic dogs are placed in a low-adsorption culture plate for amplification to form embryoid bodies, then the embryoid bodies are placed in a gelatin-coated culture plate, the induction liquid is replaced, and then the mesenchymal stem cells are obtained through induction. The induction method disclosed by the invention is simple, and the induced mesenchymal stem cells have adipogenic and osteogenic differentiation capabilities.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and in particular relates to an induction solution and an induction method for converting canine induced pluripotent stem cells into mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to self-renew and differentiate in multiple directions. They are found in various tissues, including bone marrow, umbilical cord, fat, and placenta. MSCs have achieved promising results in the clinical treatment of cardiovascular and cerebrovascular diseases, spinal cord injury, diabetes, and liver cirrhosis. Their robust proliferation capacity, multidirectional differentiation potential, and immunomodulatory properties hold great promise for their clinical application in cell therapy.

[0003] Primary mesenchymal stem cells have the advantages of a wide range of sources, ease of isolation, rapid culture, and rapid expansion. However, the biological effects of mesenchymal stem cells from different donors and tissue sources vary, the preparation process is difficult to control, and there is a potential risk of tumor cell contamination. These factors have hindered the large-scale preparation and clinical application of primary mesenchymal stem cells. Therefore, mesenchymal stem cells with a stable source and high homogeneity will be a research hotspot for future cell therapy and its clinical application.

[0004] Induced pluripotent stem cells (iPSCs) are cells that have been differentiated and reprogrammed by introducing exogenous genes, thereby obtaining cells with stem cell characteristics and unlimited proliferation capacity. Therefore, there are no ethical issues involved, and the source of pluripotent stem cells is stable, which has little impact on experimental results and can be industrially prepared. However, there are still some technical defects in inducing iPSCs into MSCs, such as the long induction time and cumbersome process, which leads to large differences between batches and affects cell stability; low induction efficiency leads to limited differentiation and self-renewal capabilities of induced cells; insufficient optimization of induction conditions leads to a low success rate; the safety and effectiveness of induced cells in clinical treatment remain to be evaluated. Therefore, more research is needed to solve the above problems, optimize technical conditions, obtain efficient and stable induced mesenchymal stem cells, and promote their application in translational medicine. Summary of the Invention

[0005] The present invention provides an induction solution and an induction method for converting canine induced pluripotent stem cells into mesenchymal stem cells. The induced mesenchymal stem cells have the ability to differentiate into adipogenesis and osteoblastogenesis, providing new ideal cells for stem cell therapy.

[0006] In the present invention, percentages are all percentages by volume, for example, 5% to 15% FBS refers to FBS with a volume percentage of 5% to 15%.

[0007] The invention provides an induction solution for converting canine induced pluripotent stem cells into mesenchymal stem cells. The induction solution comprises a basal culture medium and 5% to 15% FBS, wherein the basal culture medium is a DMEM / L culture medium.

[0008] In a preferred embodiment of the present invention, the induction solution further comprises 1% to 2% 100 mM sodium pyruvate solution, 1% to 2% 100× non-essential amino acid solution, 1% to 2% 200 mM glutamine cell culture additive and 10 to 25 ng / ml fibroblast growth factor.

[0009] In a preferred embodiment of the present invention, the induction solution includes 10% FBS;

[0010] The induction solution also includes: 1% 100mM sodium pyruvate solution, 1% 100× non-essential amino acid solution, 1% 200mM glutamine cell culture additive and 20ng / ml fibroblast growth factor.

[0011] The present invention also provides the use of the induction solution in inducing canine induced pluripotent stem cells to obtain mesenchymal stem cells.

[0012] The present invention also provides a method for inducing the transformation of canine induced pluripotent stem cells into mesenchymal stem cells, comprising the following steps: placing the canine induced pluripotent stem cells in a low-adsorption culture plate and amplifying them to form embryoid bodies;

[0013] The embryoid bodies are placed in a gelatin-coated culture plate and induced and cultured using the induction solution to obtain mesenchymal stem cells.

[0014] In a preferred embodiment of the present invention, the culture medium used for amplification includes mTeSR complete culture medium.

[0015] In a preferred embodiment of the present invention, the gelatin coating content of the culture plate is 0.2% by volume.

[0016] In a preferred embodiment of the present invention, the embryoid bodies are grown at a rate of 5×10 4 The cells were plated onto the gelatin-coated culture plates.

[0017] In a preferred embodiment of the present invention, the induction solution is added after the cells in the gelatin-coated culture plate have adhered to the wall.

[0018] In a preferred embodiment of the present invention, the induction medium is replaced every 48 hours, and the induction culture is continuously carried out for more than 14 days.

[0019] Beneficial Effects: The present invention provides an induction medium for converting canine induced pluripotent stem cells into mesenchymal stem cells. The induction medium comprises DMEM / L medium as a basal medium and 5% to 15% FBS. The present invention also provides a method for inducing canine induced pluripotent stem cells into mesenchymal stem cells using the induction medium. The canine induced pluripotent stem cells are expanded in a low-adsorption culture plate to form embryoid bodies, which are then placed in a gelatin-coated culture plate. The induction medium is then replaced to induce mesenchymal stem cells.

[0020] The present invention utilizes induced pluripotent stem cells to obtain mesenchymal stem cells, replacing the method of obtaining samples from living animals, avoiding ethical issues, reducing the use of experimental animals, and reducing experimental costs to a certain extent. The induction method described in the present invention is simple, and the induced mesenchymal stem cells have the ability to differentiate into both adipogenic and osteogenic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The cell morphology after induction for different time periods;

[0022] Figure 2 Figures 1 and 2 show the comparison of pluripotent gene expression before and after induction. Figure A shows the mRNA expression of pluripotent genes in canine induced pluripotent stem cells after 35 days of differentiation into mesenchymal stem cells, and Figure B shows the protein expression of pluripotent genes in canine induced pluripotent stem cells after 35 days of differentiation into mesenchymal stem cells.

[0023] Figure 3 Figures A and B show the mRNA expression of mesenchymal stem cell marker genes after 35 days of differentiation of canine induced pluripotent stem cells into mesenchymal stem cells. Figures C and D show the protein expression of mesenchymal stem cell marker genes after 35 days of differentiation of canine induced pluripotent stem cells into mesenchymal stem cells.

[0024] Figure 4 This is the result of CD29 immunofluorescence of mesenchymal stem cells after induction;

[0025] Figure 5 This is the result of CD44 immunofluorescence of mesenchymal stem cells after induction;

[0026] Figure 6 This is the result of CD90 immunofluorescence of mesenchymal stem cells after induction;

[0027] Figure 7 This is the result of CD105 immunofluorescence of mesenchymal stem cells after induction;

[0028] Figure 8 This is the result of CD34 immunofluorescence of mesenchymal stem cells after induction;

[0029] Figure 9 This is the result of CD45 immunofluorescence of mesenchymal stem cells after induction;

[0030] Figure 10 This is the staining result of dog induced adipogenic differentiation of mesenchymal stem cells;

[0031] Figure 11 This is the staining result of inducing osteogenic differentiation of mesenchymal stem cells in domestic dogs;

[0032] Figure 12 This is a comparison of marker gene expression between canine induced mesenchymal stem cells and canine umbilical cord mesenchymal stem cells;

[0033] Figure 13 This is a comparison chart of the activity of domestic dog induced mesenchymal stem cells and domestic dog umbilical cord mesenchymal stem cells. DETAILED DESCRIPTION

[0034] The invention provides an induction solution for converting canine induced pluripotent stem cells into mesenchymal stem cells. The induction solution comprises a basal culture medium and 5% to 15% FBS, wherein the basal culture medium is a DMEM / L culture medium.

[0035] The volume percentage of FBS in the induction solution of the present invention can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Of course, the percentage can also be expressed as a decimal point between any two values, and the same applies hereinafter. In one embodiment of the present invention, 10% FBS is used as an example for description.

[0036] The induction solution of the present invention also includes a sodium pyruvate solution (Sodium Pyruvate Solution), and when the Sodium Pyruvate Solution is 100mM, its volume percentage is 1% to 2%. In one embodiment, 1% 100mM Sodium Pyruvate Solution is used as an example for description.

[0037] The induction solution of the present invention also includes a non-essential amino acid solution (MEM Non-Essential Amino Acids Solution). When the MEM Non-Essential Amino Acids Solution is 100×, its volume percentage is 1% to 2%. In one embodiment, 1% 100× MEM Non-Essential Amino Acids Solution is used as an example for description.

[0038] The induction solution of the present invention also includes glutamine cell culture additive (GlutaMAX TMSupplement), and the GlutaMAX TM When the Supplement is 200mM, its volume percentage is 1% to 2%. For example, in one embodiment, 1% GlutaMAX TM Supplement is used as an example to illustrate.

[0039] The induction solution of the present invention also includes fibroblast growth factor (FGF), and the concentration of the FGF is 10-25 ng / ml. In one embodiment, 20 ng / ml FGF is used as an example for illustration.

[0040] In the present invention, sodium pyruvate solution is added to the cell culture medium as a carbon source, which can promote cell growth to a certain extent; non-essential amino acid solution is used as an additive to the cell culture medium to improve cell growth and activity; glutamine cell culture additive has good stability, significantly reduces the accumulation of toxic ammonia, and improves cell viability and growth; fibroblast growth factor (FGF) is a key component for maintaining cultured stem cells in an undifferentiated state.

[0041] The present invention does not specifically limit the sources of the components in the induction solution, such as DMEM / L culture medium, FBS, 100mM Sodium Pyruvate Solution, 100×MEM Non-Essential Amino Acids Solution, GlutaMAX TM Supplement was purchased from Gibco; FGF was purchased from Peprotech.

[0042] The present invention also provides the use of the induction solution in inducing canine induced pluripotent stem cells to obtain mesenchymal stem cells.

[0043] The present invention also provides a method for inducing the transformation of canine induced pluripotent stem cells into mesenchymal stem cells, comprising the following steps: placing the canine induced pluripotent stem cells in a low-adsorption culture plate and amplifying them to form embryoid bodies;

[0044] The embryoid bodies are placed in a gelatin-coated culture plate and induced and cultured using the induction solution to obtain mesenchymal stem cells.

[0045] The canine induced pluripotent stem cells described in the present invention can be frozen canine induced pluripotent stem cells taken out from liquid nitrogen. The frozen canine induced pluripotent stem cells are quickly thawed in a 37°C water bath and then centrifuged at 1000 rpm for 3 minutes. The cryogen is removed, the cells are resuspended in mTeSR complete culture medium, and cultured in a low-adsorption culture plate.

[0046] Each 50 mL of the mTeSR complete culture medium of the present invention includes: 39.84 ml mTeSR TM 1 basal medium, 10 ml 5×Supplement, 10 ng / ml FGF, 10 ng / ml hLIF, 0.3 μM Pifithrin-μ, 0.5 μM A83-01, 0.5 μM Y27632, 0.5 μM PD0325901, 3 μM CHIR99021 and 250 μM Sodium Butyrate, among which mTeSR pluripotent stem cell medium was purchased from STEMCELL, hLIF was purchased from Peprotech; A83-01, Y27632, CHIR99021 were purchased from MCE; Pifithrin-μ, CHIR99021, Sodium Butyrate were purchased from Selleck.

[0047] The culture of the present invention includes culturing in the mTeSR complete culture medium, the low adsorption culture time is 48 hours, and the culture temperature is 37°C.

[0048] The present invention arranges the embryoid bodies onto a gelatin-coated culture plate, wherein the gelatin coating of the culture plate has a mass volume percentage of 0.2%, and the embryoid bodies are arranged onto the gelatin-coated culture plate. The present invention adds the induction solution to the gelatin-coated culture plate after the cells adhere. The present invention performs culture under the aforementioned conditions, and after 24 hours of cell adherence, adds the induction solution to induce transformation, with the solution being changed every 48 hours for a continuous induction period of at least 14 days. The gelatin described in the examples of the present invention was purchased from Sigma.

[0049] In the embodiment of the present invention, mesenchymal stem cell induction fluid 1 and mesenchymal stem cell induction fluid 2 were constructed. After 14 days of induction, spindle-shaped and fibroblast-like cell populations gradually appeared in the mesenchymal stem cell induction fluid 1 and the mesenchymal stem cell induction fluid 2, and were basically stable after 21 days.

[0050] Mesenchymal stem cell induction medium 1: 45 ml DMEM / L, 5 ml FBS;

[0051] Mesenchymal stem cell induction solution 2: 45ml DMEM / L, 5ml FBS, 0.5ml 100mM Sodium Pyruvate Solution, 0.5ml 100×MEM Non-Essential Amino Acids Solution, 0.5ml GlutaMAX TMSupplement and 20ng / ml FGF. Using the induction method of the present invention, the mesenchymal stem cells induced have the ability to differentiate into adipogenesis and osteoblastogenesis, providing new ideal cells for stem cell therapy.

[0052] To further illustrate the present invention, the induction solution and induction method for converting canine induced pluripotent stem cells into mesenchymal stem cells provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1: Transformation of Canis lupus induced pluripotent stem cells into mesenchymal stem cells

[0054] Canine induced pluripotent stem cells were taken out of liquid nitrogen, thawed quickly in a 37°C water bath, and centrifuged at 1000 rpm for 3 min. The cryogen was removed and the cells were resuspended in mTeSR complete culture medium and cultured in low-adhesion culture plates.

[0055] The expanded canine induced pluripotent stem cells were cultured at a rate of 5×10 4 Plate the cells onto a 12-well plate coated with 0.2% gelatin. After 24 hours, the cells adhered to the wall and were induced with mesenchymal stem cell induction solution 1, mesenchymal stem cell induction solution 2, and mesenchymal stem cell induction solution 3, respectively. The solution was changed every 48 hours for 35 consecutive days. Figure 1 shown.

[0056] In this embodiment, the reagents are prepared as follows:

[0057] mTeSR complete medium: 39.84 ml mTeSR TM 1 basal medium, 10 ml 5× Supplement, 10 ng / ml FGF, 10 ng / ml hLIF, 0.3 μM Pifithrin-μ, 0.5 μM A83-01, 0.5 μM Y27632, 0.5 μM PD0325901, 3 μM CHIR99021 and 250 μM Sodium Butyrate.

[0058] Mesenchymal stem cell induction medium 1: 45 ml DMEM / L and 5 ml FBS.

[0059] Mesenchymal stem cell induction solution 2: 45ml DMEM / L, 5ml FBS, 0.5ml 100mM Sodium Pyruvate Solution, 0.5ml 100×MEM Non-Essential Amino Acids Solution, 0.5ml GlutaMAX TM Supplement and 20ng / ml FGF.

[0060] Mesenchymal stem cell induction medium 3: 45 ml DMEM / L and 5 ml KSR.

[0061] The results showed that spindle-shaped and fibroblast-like cell populations gradually appeared in mesenchymal stem cell induction fluid 1 and mesenchymal stem cell induction fluid 2 after 14 days of induction, and were basically stable after 21 days, but mesenchymal stem cell induction fluid 3 did not induce successfully.

[0062] Example 2: Detection of mRNA expression of relevant marker genes before and after induction

[0063] In this example, the RNA extraction kit was purchased from Quanshijin Company; the reverse transcription kit and SYBR select MasterMix were purchased from Novozymes.

[0064] The resuscitated canine induced pluripotent stem cells and mesenchymal stem cells derived from induction solution 1 and induction solution 2 after 35 days were obtained from Example 1. Total RNA was extracted, and the results were obtained by reverse transcription and quantitative fluorescence PCR. Figure 2 The expression of pluripotent stemness genes OCT4, Sox2, and Nanog mRNA is shown in A; Figure 3 The positive marker genes of mesenchymal stem cells shown in A are CD29, CD44, CD90, and CD105; Figure 3 The expression of hematopoietic stem cell marker genes CD34 and CD45 mRNA is shown in B.

[0065] Table 1 Fluorescence quantitative PCR primer sequence information

[0066]

[0067]

[0068] 20 μL fluorescence quantitative PCR reaction system: cDNA 2 μL, Forward Primer (10 μM) 0.8 μL, Reverse Primer (10 μM) 0.8 μL, SYBR select Master Mix 10 μL and RNase-free ddH2O 6.4 μL.

[0069] Fluorescence quantitative PCR program: 50°C for 2 min; 95°C for 5 min; 95°C for 15 s, 60°C for 1 min, 40 cycles; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

[0070] The results showed that 35 days after canine induced pluripotent stem cells differentiated into mesenchymal stem cells, the expression of the pluripotent gene OCT4 mRNA in mesenchymal stem cells transformed by induction medium 1 decreased by 4.3 times, and the expression of the pluripotent gene OCT4 mRNA in mesenchymal stem cells transformed by induction medium 2 decreased by 5.5 times compared with induced pluripotent stem cells; the expression of the pluripotent gene Sox2 mRNA in mesenchymal stem cells transformed by induction medium 1 decreased by 29.4 times, and the expression of the pluripotent gene Sox2 mRNA in mesenchymal stem cells transformed by induction medium 2 decreased by 13.9 times; the expression of the pluripotent gene Nanog mRNA in mesenchymal stem cells transformed by induction medium 1 decreased by 3.4 times, and the expression of the pluripotent gene Nanog mRNA in mesenchymal stem cells transformed by induction medium 2 decreased by 4.2 times.

[0071] Compared with induced pluripotent stem cells, the expression level of CD29 mRNA in mesenchymal stem cells transformed by induction medium 1 increased by 2.3 times, and the expression level of CD29 mRNA in mesenchymal stem cells transformed by induction medium 2 increased by 2.1 times; the expression level of CD44 mRNA in mesenchymal stem cells transformed by induction medium 1 increased by 1.3 times, and the expression level of CD44 mRNA in mesenchymal stem cells transformed by induction medium 2 increased by 1.9 times; the expression level of CD105 mRNA in mesenchymal stem cells transformed by induction medium 1 increased by 1.8 times, and the expression level of CD105 mRNA in mesenchymal stem cells transformed by induction medium 2 increased by 2.3 times; however, the expression level of CD90 mRNA in mesenchymal stem cells transformed by induction mediums 1 and 2 was significantly decreased. Compared with induced pluripotent stem cells, the expression level of CD34 mRNA, a hematopoietic stem cell marker, in mesenchymal stem cells transformed by induction medium 1 increased by 6.3 times, and the expression level of CD34 mRNA in mesenchymal stem cells transformed by induction medium 2 increased by 6.5 times; the expression level of CD45 mRNA in mesenchymal stem cells transformed by induction medium 1 increased by 10.9 times, and the expression level of CD45 mRNA in mesenchymal stem cells transformed by induction medium 2 increased by 9.9 times.

[0072] Example 3: Detection of related marker gene protein expression levels before and after induction

[0073] In this example, RAPI protein lysate and BCA protein concentration detection kit were purchased from Thermo Fisher; One-Step PAGE GelFast Preparation Kit (10%) was purchased from Novozymes; OCT4, Sox2, and Nanog were purchased from Santa Cruz; CD29, CD44, CD90, CD105, CD34, and CD45 were purchased from abcam; GAPDH was purchased from ABclonal; Anti-rabbit IgG, HRP-linked Antibody and Anti-mouse IgG, HRP-linked Antibody were purchased from CST; and chemiluminescence reagents were purchased from Beyotime.

[0074] Using the same method as in Example 1, mesenchymal stem cells were obtained after 35 days of induction with Induction Solution 1 and Induction Solution 2. An appropriate amount of RAPI protein lysis buffer was added, and the cells were lysed at 4°C for 30 minutes. The cells were centrifuged at 14,000 g for 15 minutes, and the supernatant was collected to obtain total protein. The protein concentration of the samples was determined using a BCA protein concentration assay kit.

[0075] 20 μg of protein was added to 10% SDS-PAGE gel for electrophoresis. After electrophoresis, the protein on the gel was transferred to PVDF membrane. The PVDF membrane was incubated with antibodies and chemiluminescence imaging was performed. Figure 2 The expression results of pluripotent gene proteins before and after induction are shown in Figure B. Figure 3 The expression results of mesenchymal stem cell marker gene proteins before and after induction are shown in C. Figure 3 Middle D shows the expression results of hematopoietic stem cell marker gene proteins before and after induction.

[0076] The results showed that 35 days after canine induced pluripotent stem cells differentiated into mesenchymal stem cells, the protein expression of the pluripotent gene OCT4 in the mesenchymal stem cells transformed by induction medium 1 decreased by 6.9 times, and the protein expression of the pluripotent gene OCT4 in the mesenchymal stem cells transformed by induction medium 2 decreased by 12.8 times compared with induced pluripotent stem cells; the protein expression of the pluripotent gene Sox2 in the mesenchymal stem cells transformed by induction medium 1 decreased by 13.9 times, and the protein expression of the pluripotent gene Sox2 in the mesenchymal stem cells transformed by induction medium 2 decreased by 8.1 times; the protein expression of the pluripotent gene Nanog in the mesenchymal stem cells transformed by induction medium 1 decreased by 16.4 times, and the protein expression of the pluripotent gene Nanog in the mesenchymal stem cells transformed by induction medium 2 decreased by 23.9 times.

[0077] Compared with induced pluripotent stem cells, the protein expression level of CD29 in mesenchymal stem cells transformed by induction medium 1 increased by 3.6 times, and the protein expression level of CD29 in mesenchymal stem cells transformed by induction medium 2 increased by 3.8 times; the protein expression level of CD44 in mesenchymal stem cells transformed by induction medium 1 increased by 1.5 times, and the protein expression level of CD44 in mesenchymal stem cells transformed by induction medium 2 increased by 2.6 times; the protein expression level of CD105 in mesenchymal stem cells transformed by induction medium 1 increased by 2.8 times, and the protein expression level of CD105 in mesenchymal stem cells transformed by induction medium 2 increased by 3.4 times; however, the protein expression levels of CD90 in mesenchymal stem cells transformed by induction mediums 1 and 2 were significantly decreased.

[0078] Compared with induced pluripotent stem cells, the protein expression level of the hematopoietic stem marker CD34 in mesenchymal stem cells transformed by induction medium 1 increased by 12.7 times, and the protein expression level of the hematopoietic stem marker CD34 in mesenchymal stem cells transformed by induction medium 2 increased by 23.7 times; there was no significant difference in the protein expression level of the hematopoietic stem marker CD45 in mesenchymal stem cells transformed by induction medium 1, and the protein expression level of the hematopoietic stem marker CD45 in mesenchymal stem cells transformed by induction medium 2 decreased by 1.7 times.

[0079] Table 2 Antibody information

[0080] Antibody Dilution ratio OCT4 1:1500 Sox2 1:1500 Nanog 1:1500 CD29 1:2000 CD44 1:2000 CD90 1:2000 CD105 1:2000 CD34 1:2000 CD45 1:2000 GAPDH 1:2000 Anti-RabbitIgG,HRP-linkedAntibody 1:2000 Anti-MouseIgG,HRP-linkedAntibody 1:2000

[0081] Example 4: Immunofluorescence detection of mesenchymal stem cells

[0082] In this example, the cell immunofluorescence fixative, permeabilization solution, blocking solution, primary antibody diluent, secondary antibody diluent, and DAPI were purchased from Beyotime; FITC-conjugated Goat anti-Rabbit IgG (H+L) and Cy3-conjugated Goat anti-Mouse IgG (H+L) were purchased from ABclonal.

[0083] Mesenchymal stem cells from Example 1, induced for 35 days with Induction Solution 1 and Induction Solution 2, were plated in 24-well plates. After confluency reached approximately 70-80%, the cells were fixed, permeabilized, and blocked. Following blocking, the cells were incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody at room temperature in the dark for 60 minutes, and with DAPI for 60 minutes in the dark.

[0084] Observe and take pictures under an inverted fluorescence microscope to obtain Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 and Figure 9The cell immunofluorescence results shown in the figure show that after 35 days of differentiation of canine induced pluripotent stem cells into mesenchymal stem cells, the mesenchymal stem cells obtained from induction medium 1 and induction medium 2 both positively expressed mesenchymal stem cell markers CD29, CD44, CD90 and CD105, but also positively expressed hematopoietic stem cell markers CD34 and CD45, and the expression level of cells derived from induction medium 2 was higher than that of cells derived from induction medium 1.

[0085] Table 3 Antibody information

[0086] Antibody Dilution ratio CD29 1:150 CD44 1:150 CD90 1:150 CD105 1:150 CD34 1:150 CD45 1:150 FITC-conjugated Goat anti-Rabbit IgG(H+L) 1:200 Cy3-conjugated Goat anti-Mouse IgG(H+L) 1:200

[0087] Example 5: Inducing adipogenic and osteogenic differentiation of mesenchymal stem cells

[0088] In this embodiment, adipogenic differentiation medium was purchased from Saiye Biotechnology Co., Ltd.; cell-specific Oil Red O staining solution was purchased from Solebold Co., Ltd.; osteogenic differentiation medium was purchased from Saiye Biotechnology Co., Ltd.; and Alizarin Red staining solution was purchased from Solebold Co., Ltd.

[0089] 1. Adipogenic differentiation of mesenchymal stem cells and red oil O staining

[0090] The mesenchymal stem cells from Example 1 were induced and transformed with induction solution 1 and induction solution 2 for 35 days, and 2×10 4 / ml were plated in 6-well culture plates coated with 0.2% gelatin, and DMEM / L complete culture medium was added. The medium was changed every 48 hours until the cell density reached 100%.

[0091] Complete culture medium was discarded and adipogenic differentiation induction medium A was added; after 3 days of induction, the medium was changed to adipogenic differentiation induction medium B; after 1 day of induction, the medium was changed back to A for induction; after 4 alternating inductions with medium A and B (16 days), the culture was maintained with medium B for 5 days. During the maintenance period with medium B, the medium was changed every 2 days.

[0092] After the induction of differentiation, the cells were fixed with ORO Fixative for 30 minutes, washed with 60% isopropanol for 5 minutes, and stained with 1 ml of Oil Red O working solution (Oil Red O stock solution: distilled water = 3:2) for 20 minutes.

[0093] Observe and take pictures under a microscope to obtain Figure 10 The adipogenic differentiation results shown in the figure show that the mesenchymal stem cells obtained by induction solutions 1 and 2 have the ability to differentiate into adipocytes.

[0094] Adipogenic differentiation induction medium A: 43.75 ml adipogenic differentiation basal medium A, 5 ml adipogenic differentiation-specific fetal bovine serum, 500 μL double-stranded antibody, 500 μL glutamine, 100 μL insulin, 50 μL 3-isobutyl-1-methylxanthine, 50 μL rosiglitazone, and 50 μL dexamethasone;

[0095] Adipogenic differentiation induction medium B: 43.9 ml adipogenic differentiation basal medium B, 5 ml adipogenic differentiation-specific fetal bovine serum, 500 μL double antibody, 500 μL glutamine and 100 μL insulin.

[0096] 2. Osteogenic differentiation of mesenchymal stem cells and Alizarin red staining

[0097] The mesenchymal stem cells from Example 1 were induced and transformed with induction solution 1 and induction solution 2 for 35 days, and 2×10 4 Plate cells at 100 μl / ml in a 6-well culture plate coated with 0.2% gelatin and add DMEM / L complete culture medium. Change the medium every 48 hours until the cell density reaches 60%. Discard the complete culture medium and add 2 ml of osteogenic differentiation induction medium. Replace the medium with fresh one every 2 days for 21 days.

[0098] After the induction of differentiation, the differentiation medium was discarded and fixed with 4% paraformaldehyde for 30 minutes; then stained with 1 ml of alizarin red solution for 20 minutes. Figure 11 The results of osteogenic differentiation shown in the figure show that the mesenchymal stem cells obtained by induction solutions 1 and 2 have the ability to differentiate into osteoblasts.

[0099] Osteogenic differentiation induction medium: 43.35 ml osteogenic differentiation basal medium, 5 ml osteogenic differentiation-specific fetal bovine serum, 500 μL double-antibody, 500 μL glutamine, 100 μL ascorbic acid, 500 μL sodium β-glycerophosphate and 5 μL dexamethasone.

[0100] Example 6: Detection of marker gene expression in induced mesenchymal stem cells and canine umbilical cord mesenchymal stem cells

[0101] Mesenchymal stem cells derived from 35-day induction solution 2 from Example 1 were used as the experimental group. Canine umbilical cord mesenchymal stem cells (ZL202010976166.2) from Zhang Yaping's group at the Kunming Institute of Zoology were used as the positive control group. The expression of mesenchymal stem cell marker genes in the two groups was compared. The detection process was consistent with that in Example 2.

[0102] The results are as follows Figure 12 As shown in the figure, compared with domestic dog umbilical cord mesenchymal stem cells, the expression levels of induced mesenchymal stem cell marker genes CD29, CD44 and CD105 mRNA were relatively high, but the expression level of CD90 mRNA was relatively low.

[0103] Example 7: Viability Detection of Induced Mesenchymal Stem Cells and Canine Umbilical Cord Mesenchymal Stem Cells

[0104] In this example, CCK8 reagent was purchased from Beyotime Pharmaceuticals.

[0105] Mesenchymal stem cells derived from induction solution 2 for 35 days in Example 1 were used as the experimental group, and canine umbilical cord mesenchymal stem cells (ZL202010976166.2) from Zhang Yaping's group at Kunming Institute of Zoology were used as the positive control group. 5 The cells were plated in 96-well culture plates, and CCK8 reagent was added after 24 hours of culture and incubated for 1 hour. The OD value was detected at a wavelength of 450 nm to compare the proliferation of mesenchymal stem cells in the two groups.

[0106] The results are as follows Figure 13 As shown in the figure, the induced mesenchymal stem cells have good activity and no significant difference from the umbilical cord mesenchymal stem cells of domestic dogs.

[0107] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An induction solution for converting canine induced pluripotent stem cells into mesenchymal stem cells, characterized in that: The induction solution comprises a basic culture medium and FBS with a volume percentage of 5% to 15%, wherein the basic culture medium is a DMEM / L culture medium.

2. The induction solution according to claim 1, characterized in that: The induction solution also includes 1% to 2% by volume of a 100mM sodium pyruvate solution, 1% to 2% by volume of a 100× non-essential amino acid solution, 1% to 2% by volume of a 200mM glutamine cell culture additive, and 10 to 25 ng / ml of a fibroblast growth factor.

3. The induction solution according to claim 1 or 2, characterized in that: The induction solution includes 10% FBS; The induction solution also includes: 1% by volume of 100mM sodium pyruvate solution, 1% by volume of 100× non-essential amino acid solution, 1% by volume of 200mM glutamine cell culture additive and 20ng / ml of fibroblast growth factor.

4. Use of the induction solution according to any one of claims 1 to 3 in inducing canine induced pluripotent stem cells to obtain mesenchymal stem cells.

5. A method for inducing the transformation of canine induced pluripotent stem cells into mesenchymal stem cells, characterized in that: The following steps are involved: Canine induced pluripotent stem cells were placed in low-adhesion culture plates and expanded to form embryoid bodies; The embryoid bodies are placed in a gelatin-coated culture plate, and induced and cultured using the induction solution according to any one of claims 1 to 3 to obtain mesenchymal stem cells.

6. The induction method according to claim 5, characterized in that: The culture medium used for the expansion includes mTeSR complete culture medium.

7. The induction method according to claim 5, characterized in that: The gelatin coating content of the culture plate is 0.2% by volume.

8. The induction method according to claim 7, characterized in that: The embryoid bodies were plated at 5×10 4 The cells were plated onto the gelatin-coated culture plates.

9. The induction method according to claim 5 or 7, characterized in that: After the cells in the gelatin-coated culture plate adhere to the wall, the induction solution is added.

10. The induction method according to claim 9, characterized in that: The induction medium was replaced every 48 hours, and the induction culture was continued for more than 14 days.

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