TWI437097B - Fluorescent protein-expressing swine bone marrow mesenchymal stem cells - Google Patents

Description

Porcine bone marrow mesenchymal stem cells

The present invention provides a porcine bone marrow mesenchymal stem cell which can express fluorescent protein, and can be used as an experimental tool for clinical cell therapy or gene therapy.

The bone marrow contains some stem cells that have the ability to differentiate into a variety of tissues, called mesenchymal or stromal stem cells, which are colony forming unit-fibroblasts (CFU-F). It is in a dormant state in the body and can enter the cell cycle by appropriate stimulation in vitro. Mesenchymal stem cells have a variety of potentials, making these cells useful as tools for clinical cell therapy or gene therapy.

Human adult stem cells have many clinical applications in clinical treatment of diseases, such as bone marrow transplantation for the treatment of blood and immune system diseases and reconstruction of the overall hematopoietic and immune system, allogeneic or xenogenic mesenchymal stem cells of patients undergoing cancer chemotherapy. Local injection or blood circulation for myocardial infarction, acute kidney injury, skin wounds, mucopolysaccharidosis, aplastic anemia, graft-vs-host disease (GVHD), diabetes, brain Treatment or improvement of diseases such as ischemic stroke, congenital diseases of the fetus in the mother's abdomen such as muscular dystrophy or osteogenesis imperfecta.

In addition to being a direct therapeutic agent for disease, mesenchymal stem cells can also be used in gene therapy, in which an adenovirus or retrovirus is used to transfect a gene to be carried into the genome of a mesenchymal stem cell, thereby migrating to a specific tissue or organ. Characteristics to treat cancer or hereditary diseases. For example, interferon beta is transfected into human stem cells and implanted in a malignant tumor to inhibit the growth of cancer cells; genetically modified cells are fused with mesenchymal stem cells, and then implanted. Repair and treatment in the body tissue, or as a transmission tool for therapeutic protein therapeutic agents, by transferring the characteristics of the body to a specific location in the body, transferring the protein drug that is easily destroyed by the immune system in the body to the action site, thereby increasing Its efficacy.

Pigs are experimental animals that are most suitable for human biomedical research models, except for non-human primates. However, in the study of pig adult stem cells, it is often difficult to obtain clear evidence to confirm that stem cells have undergone induced differentiation and transplantation. New tissue is indeed produced by differentiation of exogenous stem cells. In addition, the problems often faced by the application of adult stem cells in regenerative medicine include the changes in the expression of fluorescent proteins, the insufficient number of cell transplantation treatments, and the differentiation potential of cells after transfection of fluorescent reporters and subsequent subcultures. And the adult stem cells after gene transfection are reported to have inconsistent fluorescence protein expression after three to five amplifications, and transient expression is observed. Even if some cells continue to express fluorescent protein, their differentiation potential will follow. The number of cell expansions increases and decreases, and the effect of cell treatment by stem cells and the number of cells that can be treated or transplanted are greatly limited.

It is an object of the present invention to provide a porcine bone marrow mesenchymal stem cell which can express fluorescent protein as a tool for human reproductive medicine research.

In one aspect, the present invention provides a porcine bone marrow mesenchymal stem cell which can express a fluorescent protein, which is inserted into a chromosome of a prokaryotic nucleus of an early porcine embryo and is subjected to embryo displacement to produce a gene which expresses a systemic fluorescent protein. The transgenic pigs were then transfected into the bone marrow of the porcine fluorescent pigs, and the normal and continuous fluorescent protein-forming adult stem cells were isolated and further purified to obtain the mesenchymal stem cells.

Another aspect of the present invention provides an experimental platform system for utilizing the porcine bone marrow mesenchymal stem cells of the fluorescent protein of the present invention for use in regenerative medicine research.

In one embodiment of the invention, the fluorescent protein is a green fluorescent protein.

The present invention provides a porcine bone marrow mesenchymal stem cell which can express fluorescent protein. In order to overcome the difficulty in confirming whether the new tissue is indeed formed by the differentiation of exogenous stem cells in the existing regenerative medicine research platform, the cells are transfected with fluorescent reporter genes to distinguish them. However, in general, fluorescently-reported gene-transfected cells and subsequent subcultured cells often fail to stably express fluorescent proteins, and the number of transfected cells is limited, and the experiment is often subject to such shortcomings, using the transgenic genes that the inventors have established. Fluorescent pig technology further develops porcine bone marrow mesenchymal stem cells that can express fluorescent protein, and can continuously provide a large amount of biological tissue materials for regenerative medical research experiments.

According to the present invention, the fluorescent reporter gene can be inserted into the chromosome of the prokaryotic nucleus of the early porcine embryo according to a known standard procedure or technique for gene transfer, and the embryo is displaced to produce a gene-transgenic gene for the expression of the fluorescent protein. pig. According to known procedures or techniques, adult stem cells which consistently and continuously express fluorescent protein are isolated from the bone marrow, and further purified to obtain mesenchymal stem cells.

The features of the present invention are more specifically described with reference to the specific embodiments of the invention which

Example 1, production of genetically-transferred pigs

The super ovulation and estrus sympathetic treatment of the pigs were tested. The new sows (gilts) of the embryos for embryos and embryos were used for sexual maturity of 6 months or older. The test embryo and the embryo-bearing sow are continuously fed for 14 to 18 days in a dose of 20 mg/day by Regument (Intervet) to achieve the simultaneous estrus treatment of the pig for the embryo and the recipient. The pigs were intramuscularly injected with pregnant horse serum (pregnant mare's serum gonadotropin, PMSG; China Chemical Pharmaceuticals) on the 14th or 18th day after feeding the media, and were given intramuscular injection 80 hours after the injection of PMSG. Human chorionic gonadotropin (hCG; Chinese chemical pharmacy), induced sputum production of superovulation; PMSG injection units of embryo and pigs were 1750 IU and 1000 IU, respectively; hCG was 1000 IU and 500 IU. The embryo was given the first artificial insemination within 28 to 30 hours after the hCG injection, and the second artificial insemination was performed 38 hours after the hCG injection. These embryos were developed to the pronuclear stage 55 hours after hCG injection; this period was selected for abdominal midline surgery, and the pig embryos were collected from the umbrella part and collected for microinjection. The collection and treatment of pronuclear embryos, the pronuclear embryos collected by surgery were placed in eppendrof containing 1 ml of DPBS, centrifuged at 12000 rpm for 10 minutes, and the fatty oil was dripped off to the side to expose the pronuclear position for profit. Gene microinjection (Figure 1). Exogenous gene for injection, DNA used for microinjection in the experiment. Escherichia coli frozen liquid containing pCX-EGFP was provided by Dr. Li Kunxiong from Taiwan Institute of Animal Science and Technology. The plastid was via the restriction enzyme Sal. After I and HindIII were cut, the only one containing cytomeglavitus immediated-early enhancer (CMV-IE enhancer), chicken beta-actin promoter (chicken beta-actin promoter), a segment of β-actin involved The sequence of the beta-actin intron, the green fluorescent protein complementary DNA (EGFP cDNA), and the length of the rabbit β-globin ploy (A) Approximately 3 kb linear DNA fragment (Fig. 2) was centrifuged at 12,000 rpm for 10 minutes before microinjection at a concentration of 1 ng/μl. Pig embryos after DNA injection, the appearance of which still retains dense yolk and complete morphology, displaces the estrus sympathetic treatment to the pronuclear stage of the embryonic sow in the second side of the fallopian tube; the number of embryos displaced by each side of the fallopian tube The average is 10~15.

Example 2: Analysis and breeding of genetically-transferred pigs

The piglets produced by the method of Example 1 were obtained by using the excitation light (optima CL-150B, Tianya) and the yellow filter to perform the preliminary screening action, and then the tissue of the ear was extracted and extracted. The genomic DNA therein is for analysis of the insertion of the foreign gene into the genomic DNA. Tissue genomic DNA was extracted and the previously obtained tissue sample was placed in 630 ul of tissue decomposing buffer [lysis buffer: 100 mM Tris‧HCl (Amresco, USA) pH 8.5, 5 mM EDT (SIGMA, USA), 200 mM NaCl (SIGMA, USA), 70 ul of 10% sodium dodecyl sulfate (SDS, MERCK, Germany), and 35 ul of 100 mg/ml proteinase K (Qiagen, Germany), after being crushed and thoroughly mixed, Place it in a 55 ° C water bath for overnight, then centrifuge at 12,000 xg for 10 min to remove hair and tissue debris, followed by equal amounts of phenol (phenol, Amresco, USA), and chloroform (chloroform, MERCK, Germany), etc. After extracting twice, the supernatant was taken by centrifugation (12000 rpm, 10 min), and 1 ml of isopropenol was added to precipitate the DNA, and finally 1 ml of 70% alcohol (absolute ethanol, MERCK, Germany) was added to wash the DNA. It was dissolved in EB buffer, and a small amount of DNA solution was taken for minicolloid electrophoresis analysis to confirm the DNA quality of the extraction. Southern-blot (Southern dot method) analysis of genomic DNA of born piglets. The probe used was to amplify the 417 fragment of EGFP cDNA sequence by PCR (2~25 ng/45 μl) and denaturing after boiling water. After 10 min on ice, a nucleic acid primer labeling set (Rediprime II random prime labeling system, Amersham Pharmacia Biotech., UK) and 50 μCi of 32p-dCTP (Amersham Pharmacia Biotech., UK) were added and cultured at 37 ° C for 1 hour. Purification kits were removed using Quick Spin Columns (Roche, Germany) to remove uncalibrated radioactivity and enzymes. The genomic DNA of each birth piglet cut by EcoR I was taken into the gelatin of the agar extract and subjected to electrophoresis treatment (depurination; 0.25 N HCl (MERCK, Germany), 15 min), to be bromophenol. After the blue dye turns from blue to yellow, the acacia gum is soaked in a denaturation solution [1.5 M NaCl (MERCK, Germany), 0.5 M NaOH (MERCK, Germany)], and the downward spray is set. Salt Bridge. The positively charged nylon membrane (Hybond-N+, Amersham Pharmacia Biotech., UK) of the transferred DNA was placed in a 2×SSC solution (Amresco, USA) for neutralization reaction and removal after 2.5 to 4 hr of transfer. The remaining acacia colloids are then directly subjected to pre-hybridazation and hybridization reactions. The pre-hybrid reaction is to first place the positively charged nylon membrane in a hybridization tube (Schotte, Germany) and add the hybrid solution before dissolution [composition: 2 × SSC, 1% SDS, 0.5%) Skim-free milk powder (Anjia, Taiwan), and 0.75/ml denatured sperm DNA (SIGMA, USA), and let it in a hybrid incubator at 62 °C (PersonalHyb) Hybrization oven, Stratagene, USA) After 2~5 h reaction, the heterozygous solution was transferred [composition: 2×SSC, 1% SDS, 0.5% skim milk powder (fat-free milk powder, Anjia, Taiwan) 0.5mg/ml denatured sperm DNA (salmon sperm DNA, SIGMA, USA) and 10% dextran sulfate (Amresco, USA), and additionally added the above-mentioned radiolabeled DNA probe and placed in 62 DNA hybridization reaction was carried out in a °C water bath for 16-24 hours. The NC membrane that completed the hybrid reaction was washed twice with a cleaning solution containing 0.1% SDS and 2 x SSC at room temperature for 10 min each time, and then a cleaning solution containing 0.1% SDS and 0.1 x SSC was used. The cells were washed twice at 55 ° C for 20 min, and finally the membrane was taken out, air-dried, and then subjected to autoradiography using an image processing system (Typhoon 9200, Amersham Bioscience, UK), and subjected to interpretation analysis by ImageQuant software.

Breeding pigs with the characteristics of green fluorescent protein are selected and raised in traditional barns, providing sufficient pig-specific diets and clean drinking water, and regularly cleaned. The pig house maintains good ventilation and fresh air.

In this example, a total of 339 pig embryos were obtained by in vivo maturation and fertilization and surgically obtained, and 278 microinjections of pig embryos in the pronuclear stage were completed, 265 of which were microinjected with pCX-EGFP exogenous gene, The embryos were placed in the 8 oviducts of the surrogate mothers. A total of 4 embryos were pregnant and 36 piglets were successfully born. The next step was to use blue light with a wavelength of 475 nm and irradiated with yellow green patches in the dark. Among the 36 piglets, 3 piglets showed green fluorescence, numbered 84-1, 64-8 and 64-9, respectively. Under natural light, the eyes, feet and nose of the three piglets were compared with normal pigs. Obvious yellow appearance. Using 15 μg of genomic DNA from 3 piglets as a genomic template, the EcoR I enzyme was used for the truncation, and the 417 fragment of the EGFP cDNA sequence was used as a probe for Southern blot analysis. The results confirmed that the three piglets did have pCX. The -EGFP gene, hereinafter referred to as fluorescent pig. At the same time, it is further observed that all the tissues and organs of the transgenic pig (fluorescent pig) producing green fluorescent protein in this embodiment express a large amount of green fluorescent protein including: rib, kidney, heart, muscle, liver, brain. , eyes, testicles, lungs, intestines, tongue, penis, as shown in Figure 3.

Example 3: Acquisition of bone marrow mesenchymal stem cells

In the present embodiment, bone marrow cells were obtained by extracting bone marrow, and purified by density gradient centrifugation (Ficoll paque).

The femur bone of the fluorescent pig selected by the method of the free example 2 was taken out, and then filtered through a 70 μm sieve, and dispensed into 5 mL per tube, and the divided bone marrow solution was centrifuged (1200 X rpm, 10 min). After that, the supernatant was removed, and 10 mL of the culture solution (αMEM, Sigma M0894 USA; 20% FBS, Hyclone USA; Pen/Str, Gibco 15140-122 USA) was added to break up the cell mass, and 5 mL of bone marrow cells were suspended. was slowly added to the centrifuge tube along with 6 mL of 0.012 ng / mL of ^{Ficoll-Paque TM pLUS (Amersham,} Sweden), was added the suspension was centrifuged good Ficoll-Paque ^TM pLUS marrow cells 1700 X rpm, 20 min. The centrifugal speed gradient starts at 400 X rpm and is adjusted up to 200 X rpm every 30 sec until 1000 X rpm. At this point, add 200 X rpm every 1 min until it reaches 1700 X rpm and continue centrifugation for 20 min. After centrifugation of the bone marrow cell suspension can be divided into four layers, from top to bottom are the plasma layer, the white turbidity and MSCs mixed leukocyte cell layer, etc., a transparent layer of Ficoll-Paque ^TM PLUS, red cell layer. The plasma layer was first aspirated, and the mixed cell layer of white turbid MSCs and white blood cells was inhaled into a 15 ml tube containing 5 ml of the culture solution. Centrifuge at 1200 X rpm for 5 min and remove the supernatant. Repeat this step twice. Finally, 2 ml of the culture solution was used to break up the cell mass and placed in a 3.5 cm culture dish for 72 hr. The suspended unattached cells are then removed, which is purified fluorescent porcine mesenchymal stem cells.

Example 4: Study on the properties of mesenchymal stem cells

So far, there has been no specific marker for detection of porcine mesenchymal stem cells, so the purity of fluorescent porcine stem cells was identified by flow cytometry using different antibodies CD4a, CD31, CD44, CD90 and CD29. Methods as below.

(1) Culture and preservation of mesenchymal stem cells

Mesenchymal stem cells are adherent cells, so when the cells proliferate to saturation (100% confluence), they must be subcultured or cryopreserved. The subculture was first removed and washed with a medium containing no fetal bovine serum to remove residual fetal bovine serum, followed by 0.25% trypsin-EDTA (Gibco 25200 USA) and placed in a 37 ° C incubator. After 5 min, the cells were rinsed off the bottom of the culture dish, and the cells were resuspended using the culture medium containing fetal bovine serum, centrifuged at 1200 X rpm for 5 min, and the supernatant was removed. Mouse MSCs were reimplanted into new culture dishes at 1:3 ratio in 1:2 or porcine MSCs. When the cells were cryopreserved, the cell pellet was suspended in a culture solution containing 10% dimethyl sulfoxide (DMSO, Sigma D2650 USA), and immediately placed in liquid nitrogen for storage.

(2) Cell count

Aspirate 50 μl of the suspension cells and mix them evenly with trypan blue (Gibco 15250-061 USA). The stained cells were dropped on a hemocytometer on a cover glass and counted under the microscope. The blue living cells are converted to represent the total number of cells.

(3) Flow cytometry - surface antigen analysis (flowcytometry)

The cultured MSCs were removed, washed twice with PBS, suspended in trypsin-EDTA, and the cell suspension was centrifuged at 1200 X rpm for 5 min. After the culture solution was aspirated, it was washed with washing buffer (49.5 mL PBS + 0.5 mL FBS), and 10 ml of washing buffer was taken to resuspend the cell block and centrifuged at 1200 X rpm for 5 min. The antibody (BD, USA) to be used for detection was mixed with cells (5 x ^{10 5} cells/100 μL). The antibodies tested were CD11b-PE, CD34-PE, CD45-PE, CD166-PE, CD133-PE, CD117-PE, CD31-PE, CD86-PE, CD29-PE, CD44-PE, MHC-II-PE. , MHC-I-PE and Sca-1-PE in leaf stem cells between mice. Leaf stem cells between CD4a-PE, CD31-PE, CD44-PE, CD90-PE, CD29-PE in pigs. The tube was wrapped in aluminum tissue and protected from light, and incubated at 4 ° C for 30 min. Then resuspend in 500 μl washing buffer and centrifuge at 2000 X rpm for 5 min. The same steps are repeated twice. The cell pellet from which the supernatant was removed was added to 500 μl of fixing buffer (48.1 mL PBS + 0.5 mL FBS + 1.35 mL formaldehyde), and then subjected to flow cytometry analysis (FACS scan flow cytometer Becton-Dickinson).

Among them, CD4a is a marker of immunoglobulin expression, and CD31 is a marker of endothelial cells, fibroblasts, platelets, phagocytic cells, T lymphocytes and natural killer cells. Therefore, leaf stem cells do not show this sign between pigs. CD44 and CD29 are markers of cell migration and attachment, and CD90 is a marker for the expression of common stem cells. Therefore, leaf stem cells between pigs show this marker. From this, it was found that purified green fluorescent bone marrow mesenchymal stem cells were indirectly confirmed by density gradient centrifugation (Ficoll paque) and confirmed by the above antibodies ( FIG. 4 ).

Example 5, Wound and Healing Assay

In vitro cell wound and healing assays were performed to determine whether fluorescent porcine mesenchymal stem cells have the ability to influence the migration and differentiation of green fluorescent genes. This method is based on the steps used by the Faber-Elman et al (1996) and Schmidt et al (2006) research teams, operating as follows: cultured mesenchymal stem cells in 10 cm culture dishes, to 90% confluence, using P 200 (200μl) tip is drawn on a line, and the migration period is measured at intervals.

The results showed that there was no significant difference in the migration and proliferation ability of leaf stem cells between green fluorescent pigs and normal pigs after 28 hours. Moreover, the ability of migration and differentiation of passage 2 and passage 13 was compared, and the results showed that cells with different number of passages did not affect their ability to migrate and proliferate. There was no significant difference in bone differentiation between normal pigs, leaf stem cells, second-generation mesenchymal stem cells with green fluorescent pigs, and thirteenth generation mesenchymal stem cells with green fluorescent pigs. This result shows that there is no difference in the ability of the fluorescent mesenchymal stem cells to proliferate and migrate in the late stage to the early stage (Fig. 5 and Fig. 6).

Example 6. Differentiation of germ layer bone, fat and cartilage in fluorescent porcine bone marrow mesenchymal stem cells

6.1 Bone marrow differentiation and detection of bone marrow mesenchymal stem cells in vitro

6.1.1. Osteogenic differentiation of bone marrow mesenchymal stem cells in vitro

The porcine bone marrow mesenchymal stem cells were cultured in a 6-well plate until 24 hr after 100% confluence, and the culture medium was removed and added to the skeletal cell-inducing medium (α-MEM + 10%). FBS + 0.1 μM dexamethasone (Sigma D8893 USA) + 10 mM glycerol-2-phosphate (Sigma G9891 USA) + 50 μM ascorbate-2-phosphate (Sigma A0207 USA), and the skeletal cell-inducing broth was changed every 3 days.

6.1.2. Alkaline phosphotase assay

The MSCs which had undergone induction of differentiation were taken out from the incubator, and the culture solution was aspirated, and the cells were thoroughly washed with PBS, and then pre-cooled methanol (-20 ° C) was added. The culture dish was transferred to a -20 ° C refrigerator for 5 min. The methanol was removed and washed with deionized water. Then FAST BCIP/NBT solution (Sigma B5655 USA) was added and incubated for 10 min at room temperature with gentle shaking. The coloring solution was aspirated and washed with deionized water, and then 300 μl of deionized water was added to keep the cells moist and quickly moved to a microscope for observation and photographing. Finally, it is preserved with gelatin + glycerol sealant.

6.1.3. Alizarin Red S calcium ion staining

The calcification of MSCs differentiated into skeletal cells was analyzed by Alizarin Red S staining. On the 21st day after differentiation, cells were fixed with 10% Formaldehyde at room temperature for 30 min, washed with PBS for 3 times and then added with 2% Alizarin Red S (Sigma). A5533 USA) solution, adjusted to pH 4.1 to 4.3 with ammonium hydroxide (Merck 8.22149 Germany) prior to use. The staining was carried out for 20 min at room temperature, and the residual stain was thoroughly washed with PBS and observed under a microscope.

6.2 In vitro fat differentiation and detection of mesenchymal stem cells

6.2.1. Induction of adipogenesis of bone marrow mesenchymal stem cells in vitro (adipogenesis)

The porcine MSCs were cultured in a 6-well plate until 24 hr after 100% confluence. The medium was removed and added to the adipocyte-inducing medium (α-MEM + 10% FBS + 1 μM). Dexamethasone (Sigma D8893 USA) + 0.5 mM isobutyl-methyxanthine (Sigma G9891 USA) + 10 μg/ml insulin () + 100 μM indomethacin, the adipocyte-inducing medium was changed every 3 days.

6.2.2. Oil Red O detection method

The MSCs which had undergone induction of differentiation were taken out from the incubator, and the culture solution was aspirated, and the cells were thoroughly washed with PBS, and then pre-cooled methanol (-20 ° C) was added. The culture dish was transferred to a -20 ° C refrigerator for 5 min. The methanol was removed and washed with deionized water. Next, the Oil Red O solution was added and stained for 15 min at room temperature, and the remaining Oil Red O was washed with PBS to observe the stained oil droplets in the cells under a microscope.

6.3. In vitro cartilage differentiation and detection of mesenchymal stem cells

6.3.1. In vitro induction of chondrocyte differentiation by mesenchymal stem cells (chondrogenesis)

The porcine MSCs were cultured in a 6-well plate until 24 hr after 100% confluence. The culture was removed and added to the chondrocyte-inducing medium (α-MEM+1% FBS+6.25). Μg/ml insulin+50 μM ascorbic acid+10 ng/ml TGF-β1 (R&D Systems, Mirmeapolis, MN, http://www.rndsystems.com), the chondrocyte-inducing culture was changed every 3 days.

6.3.2. Toluidine blue assay

The MSCs which had undergone induction of differentiation were taken out from the incubator, and the culture solution was aspirated, and the cells were thoroughly washed with PBS, and then pre-cooled methanol (-20 ° C) was added. The culture dish was transferred to a -20 ° C refrigerator for 5 min. The methanol was removed and washed with deionized water. Next, the Toluidine blue solution was added and stained for 15 min at room temperature, and the remaining Toluidine blue was washed with PBS, and the stained chondrocytes in the cells were observed under a microscope.

6.4. Results

In the in vitro differentiation assay of green fluorescent porcine bone marrow mesenchymal stem cells, the osteoblast differentiation test showed that the mesenchymal stem cells were cultured in the bone differentiation medium for three weeks, and a precipitate of calcium crystal was found, and the mesenchymal stem cells were differentiated into hard bone cells under fluorescence. Fluorescent protein was expressed. Alkaline phospatase was used to detect bone differentiation on the tenth day. ARS staining was used to detect bone differentiation. On the 21st day, bone calcium deposition was observed. In the chondrocyte differentiation assay, the mesenchymal stem cells were differentiated into chondrocytes to express luciferin under fluorescence. Detection of cartilage-differentiated tissue sections by Toluidine blue staining micromass pellet showed glycosaminoglycan expression. In the adipocyte differentiation, the leaf stem cells were cultured in the adipocyte differentiation medium for three weeks to form oil droplets, and the mesenchymal stem cells were differentiated into adipocytes to express fluorescent protein under fluorescence, and the lipid was detected by Oil Red O. The particles are in the cells. It was revealed by flow cytometry that the differentiation of the mesenchymal stem cells into osteoblasts, chondrocytes and adipocytes still showed a large amount of fluorescent protein (Fig. 7 and Fig. 8).

Example 7. Differentiation of porcine bone marrow mesenchymal stem cells across the germ layer

7.1 In vitro neural differentiation and detection of mesenchymal stem cells (ectoderm)

The fluorescent porcine MSCs were cultured in a 6-well plate until 24 hr after 100% confluence. The culture was removed and added to the neural cell-inducing medium (α-MEM + 0.5 mM isobutyl-methylxanthine). (Sigma-Aldrich) and 10 μg/ml insulin (Sigma-Aldrich)) The chondrocyte-inducing medium was changed every 3 days. After differentiation into neuron-like cells on the ninth day, cell immunostaining was performed using beta 3 tubulin.

The fluorescent porcine bone marrow mesenchymal stem cells were cultured in a neuroblast-like differentiation medium for three weeks, and the differentiated neuron-like cells continued to exhibit fluorescent protein. Such neuronal cells were found to exhibit beta 3 tubulin by cellular immunostaining (Fig. 9).

Differentiation and detection of in vitro insulin secretion cells in 7.2 mesenchymal stem cells (endoderm)

After ⁶ ╳ ^{10 6} of EGFP-pMSCs were obtained, they were treated with 0.25% Trypsin-EDTA (Gibco, 25200, USA). The cells were inoculated into an ultra low cluster plate (Costar, COR3471, USA) having a diameter of 3.5 cm for 24 hours. After 24 hours, the green fluorescent porcine mesenchymal stem cells in the ultra low cluster plate will form tight globular masses. The cells that formed the globular clumps were seeded in a poly-D-lysine extracellular matrix culture dish. The above can form a dense mass of fluorescent porcine bone marrow mesenchymal stem cells. This pellet was seeded in αMEM + 10 mM nicotinamide (Sigma, N0636, USA) + 10 μM exendin-4 (Sigma, E7144, USA) + 2% B27 serum-free supplement (Invitrogen, 17504-044, USA) +1 % N2 serum-free supplement (Invitrogen, 17502-048, USA) was further supplemented with glucose (Sigma, G7021, USA) to 28.5 mM to induce differentiation culture. The chondrocyte-inducing culture solution was changed every 3 days.

The purified porcine bone marrow mesenchymal stem cells were cultured and added to the induced differentiation medium, and the cell type was gradually changed on the sixth day of culture, and the differentiated cells were expressed as albumin by cell immunofluorescence staining. The specific expression of hepatocytes showed that the cells on the twelfth day after differentiation had the property of showing albumin (Fig. 10). Therefore, the potential of porcine bone marrow mesenchymal stem cells to differentiate into hepatocytes in vitro is determined, and the cells can stably express green fluorescent protein after differentiation, and thus can be subsequently applied to in vivo tracking experiments.

7.3 In vitro liver differentiation and detection of fluorescent porcine mesenchymal stem cells (endoderm) The purified porcine bone marrow mesenchymal stem cells were adjusted to a density of 5×10 ⁴ cells/cm ^{2 and} cultured in 6-well plate to hepatocytes. The differentiation-inducing medium (α-MEM+HGF (Peprotech 100-31) + FGF-4 (Peprotech 100-39)) was stimulated, and the medium was changed every three days until the 12th day after differentiation was induced.

The expression of green fluorescent protein in the differentiation of fluorescent porcine bone marrow mesenchymal stem cells into a type of insulin-secreting cells induced by cell clumps. Fluorescent porcine mesenchymal stem cells after differentiation (14 days) were expressed by cellular immunostaining to express insulin protein (Fig. 11 and Fig. 12). The expression of green fluorescent porcine mesenchymal stem cell-derived islet cell-associated transcription factors and genes after differentiation includes Insulin, Glucagon, Glucokinase, Somatostatin, Pax6, Nkx6.1, Glut2. (Fig. 13).

Example 8, gene therapy study of diabetic mice

8.1 Cellular immunostaining (Immunocytochemistry)

The cells were rinsed with phosphate-buffered saline (PBS) and fixed with 4% para-formaldehyde (Sigma, 158127, USA) for 15 minutes. After washing three times with PBS (5 minutes each time), PBST (PBS containing 0.25% Triton X-100) was added for 10 minutes, and then washed three times with PBS (5 minutes each), and the inclusion 1 was added. % BSA (Sigma, A7030, USA) PBST to prevent non-specific heterozygous antibody, and then the first-stage antibody polyclonal guinea pig anti-insulin diluted with PBST containing 1% BSA (Sigma, A7030, USA) 1:200) (Abeam, ab7842, USA) and polynucleotide guniea pig anti-C-peptide (1:200) (Abeam, ab30477, USA) were incubated for 1 hour at room temperature. Rinse three times with PBS (5 minutes each), then add the secondary antibody rodamine-conjucated anti-guinea pig IgG (1:200) (Abeam, ab6768, USA) and DAPI (1:1000) (Invitrogen, D3571, USA) was incubated at room temperature for 1 hour and then observed using a fluorescence microscope.

8.2 Reverse transcription polymerase chain reaction (RT-PCR)

RNA was extracted by TRIZOL method (Ambion, AM9738, USA), and 3 ml of TRIzol reagent (Ambion, AM9738, USA) was added to each 10 cm ² dish and mixed in a 1.5 ml plastic centrifuge tube. After mixing, it was left at room temperature for 5 minutes. Thereafter, 0.2 ml of chloroform (Merck, 1.02445.1000, USA) was added and gently shaken for 15 seconds and allowed to stand at room temperature for 3 minutes, and then centrifuged at 12,000 rpm for 15 minutes at 4 °C. After centrifugation, RNA->Protein->DNA stratification can be obtained. The supernatant (RNA) is replaced with a new 1.5 ml plastic centrifuge tube, followed by 0.5 ml Phenol (Merck, 400 ml, 0981, USA)/Chloroform. (Merck, 1.02445.1000, USA) Mixture and shake well, centrifuge at 12000 rpm for 15 minutes at 4 ° C. Repeat this step to remove the protein, then add the supernatant to a new 1.5 ml plastic centrifuge tube. The mixture was mixed with 0.5 ml of Chloroform and centrifuged at 12000 rpm for 15 minutes at 4 ° C to remove phenol (Merck, 400 ml, 0981, USA). After centrifugation, the supernatant was placed in a new 1.5 ml plastic centrifuge tube, and 0.6 ml of isopropanol (Merck, 1.09634.1000, USA) was added to precipitate the RNA, which was then left at room temperature for 10 minutes and then at 12,000 rpm. Centrifuge at 4 ° C for 20 minutes. The precipitate is the RNA pellet. 1 ml of 75% ethanol was added to the pellet and centrifuged at 12000 rpm for 5 minutes at 4 °C. The precipitate was added to 21 μl of DEPC-ddH ₂ O reconstituted RNA pellet. After re-dissolving, it was placed at 55-60 ° C for 5 minutes, and the concentration was measured with nanodrop. Take 500 μg of total RNA in an RNAse-free centrifuge tube, add 1 μl of oligo(dT)20 (50 μM), 1 μl of 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH), Add sterile deionized water to a total volume of 13 μl, heat it to 65 ° C for 5 minutes, then place on ice for 1 minute, add 4 μl of 5X First-Strand Buffer, 1 μl of 0.1 M DTT, 1 μl of RNaseOUT ^TM Recombinant RNase Inhibitor (40 units / μl ) (Invitrogen, 10777-019, USA), after mixing with SuperScript ^TM III RT 1 μl of (200 units / μl) (Invitrogen , 18080-093, USA) was placed 50 ℃, 60 minutes, 70 ° C, 15 minutes. Finally, the cDNA was stored frozen in a -20 ° C refrigerator. The total PCR reaction volume is 20 μl, containing 5.5 μl of deionized water, 1 μl of cDNA, 1 μl of Primer (10 μM) (Forward+Reverse), 1 μl of 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH) (Invitrogen, 18427-013, USA), 10X PCR Buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl) 2.0 μl, and Taq DNA polymerase (5 U/μl) (Invitrogen, 10966) -018,USA) 0.2 μl, 50 mM MgCl ₂ 0.7 μl, cDNA (from first-strand reaction) 1 μl, added sterile deionized water to a total volume of 25 μl; 32-35 cycles with ABI 9700, temperature set to 95 °C-45 seconds, 58 °C-30 seconds, 72-45 °C seconds, and finally extended by 72 °C chain for 7 minutes (Insulin, Glucagon, Pax 6, Nkx 6.1, Somatostatin, Glut2, Glucokinase, GAPDH annealing temperature are 58 °C, and the number of cycles Pax 6 is 30, the rest are 35 cycles).

8.3 Test procedure for subcutaneous transplantation of EGFP-pIPCs

NOD/LtJ diabetic mice that developed hyperglycemia after induction were first burred with a 18G needle after depilation, and the cells were placed in 500 μl bFGF (0.1 mg/ml), 1 ml syringe. Directly transplanted into the cells subcutaneously on the back, the cells include the following: 1. Insulin-secreting cells (EGFP-pIPCs) derived from green fluorescent porcine mesenchymal stem cells after differentiation (6╳10 ⁶ ) (n=6) . 2. PBS (n=3). 3. EGFP-MSCs (6╳10 ⁶ ) (n=3). 4. Control group not processed (n=6). The wound was sutured after implantation for observation on the following day.

8.4 Detection of blood sugar and detection of survival rate

In this experiment, the test mice were irradiated for 30 minutes by infrared light, the systemic circulation was increased to observe the tail vein, and then the 30 G needle was used to bend the ophthalmic clip to 90 degrees. The tail vein was injected and blood was taken, and then Roche was used (ACCU-CHEK). Blood glucose machine to detect blood sugar. It was detected every two days after STZ treatment and after cell transplantation. Its blood glucose detection range is 10~600 mg/dL. The survival rate of diabetic mice was also observed after blood glucose was detected.

8.5 Results

Fluorescent porcine bone marrow mesenchymal stem cells were seeded in a poly-D-lysine coated (MW>300,000) culture dish with high-purification medium without serum (αMEM medium+B27 supplement+N2 supplement+28.5 mM glucose). +10 mM Exedin-4+10 mM nicotinamide) began to differentiate, and cells were aggregated into islet-like clusters within 14 days. And for the molecular characteristics of insulin-like cells derived from transdifferentiation of fluorescent porcine bone marrow mesenchymal stem cells, pancreatic specific expression genes and transcription factors (Insulin, Glucagon, Somatostatin, Glucokinase, Pax6, Nkx6.1) were confirmed by RT-PCR. , Glut2) has been shown, and it has been confirmed by cell immunostaining technique that insulin-like cells can express Insulin after differentiation. This result confirms that differentiated β cells of bone marrow stem cells can replace damaged β cells and have reduced diabetes. Hyperglycemic function in rats. Therefore, using the fluorescent porcine bone marrow mesenchymal stem cells of the present invention as an experimental platform, the fluorescent porcine bone marrow mesenchymal stem cells are transdifferentiated into insulin-secreting cells and then subcutaneously colonized to verify the differentiation of the insulin-like cells in the first-type diabetic mice ( NOD/LtJ) Whether there is functional research in the body. According to the insulin-secreting cell differentiation platform of the present embodiment, a sufficient amount of insulin-secreting cells (6╳10 ⁶ ) can be obtained within 9 days, and the induction differentiation time is shortened compared with other experiments. In this experiment, fluorescent mouse porcine mesenchymal stem cells were induced to differentiate into insulin-secreting cells (6╳10 ⁶ ) subcutaneously injected into diabetic mice (six) as treatment group, and also injected with fluorescent porcine bone marrow mesenchymal stem cells (6╳10 ⁶ ) (three), PBS (three) and hyperglycemic diabetic mice served as control group (six). It is immersed in insulin-secreting cells derived from green fluorescent porcine bone marrow mesenchymal stem cells for 3 to 4 days, and the first type of diabetic mice (NOD/LtJ) with a blood glucose level close to 600 mg/dl (6) The blood sugar has a tendency to slowly decline until the blood glucose level is significantly improved compared with the control group 20 days after burial, and the survival rate is still 100% at 20 days, and the other group died at 16 to 18 days. The differentiated fluorescent porcine insulin-secreting cells have been shown to secrete insulin and can be hypografted into NOD/LtJ mice subcutaneously to reduce hyperglycemia in vivo (Figures 14 and 15).

The invention can display the fluorescent protein porcine bone marrow mesenchymal stem cells into a platform for transdifferentiating into insulin-secreting cells in vitro, and can establish a sufficient amount of insulin-secreting cells in a short time, and if the autologous stem cells used in the diabetic patients can be implemented in the future, Treating patients with diabetes itself may solve long-term dependence on insulin injection, insufficient source of islet organ/cell transplantation and immune rejection, and has great potential for clinical application in the treatment of diabetes.

Similarly, the fluorescent porcine bone marrow mesenchymal stem cells of the present invention can also be applied to osteoporosis mice, and photographs of the bone marrow washed out after 2 months of treatment are shown in Figs.

In summary, the porcine bone marrow mesenchymal stem cells of the present invention which can express fluorescent proteins can be efficiently transdifferentiated into other cells and further applied to disease animal models or human clinical treatment studies.

Figure 1 is a photograph of the development of a foreign gene injected into the pronucleus of a fertilized egg (x 200) by microscopy, (A) before microinjection of the gene, and (B) after microinjection of the gene (prokaryotic membrane expansion arrow) ).

Figure 2 is a map of the pCX-EGFP restriction enzyme.

Figure 3 shows the production of transgenic pigs with green fluorescent protein by pronuclear microinjection, (A) is the CMV-β-actin-EGFP transgenic gene; (B) is the control group under natural light. Genetically modified pigs (left) and transgenic pigs with green fluorescent protein (right), and transgenic pigs have yellow eyes, hooves and nose under natural light; (C) are born in 36 births of piglets 3 piglets showed green fluorescence in the dark field through blue excitation light; (D) in the GFP filter and blue light, the green fluorescent protein transgenic pig showed a large amount of green fluorescent protein; E) Analysis of genomic DNA of transgenic pigs using genomic DNA cut with Southern blot EcoR I with green fluorescent protein gene fragment (probe: 739 bp) M, marker; Pc, positive control; Nc, negative control. (FQ) green Fluorescent protein transfer gene All tissues and organs of the pigs express a large amount of green fluorescent protein (right picture, LY strain pig) including rib (F), kidney (G), heart (H), muscle (I), liver (J), brain (K), eye (L), testicular (M), lung (N), intestine (O), tongue (P), penis (Q). Wild-type tissue/organ ( Left picture, Li Song mini pig) As a control group.

Figure 4 shows the phenotype of fluorescent porcine bone marrow mesenchymal stem cells and their surface antigen analysis: (A) green fluorescent protein transgenic pig bone marrow mesenchymal stem cells were photographed under white light; (B) under fluorescent light; C) detecting the expression of green fluorescent protein in fluorescent porcine bone marrow mesenchymal stem cells by Western blotting; (D) detecting the expression of green fluorescent protein of bone marrow mesenchymal stem cells by flow cell migration; (E) being flow cytometry The instrument detects the surface antigen of green fluorescent protein transgenic pig mesenchymal stem cells.

Figure 5 is a test of wound and repair of different generations of fluorescent porcine bone marrow mesenchymal stem cells: there is no significant difference in migration and proliferation ability between fluorescent porcine mesenchymal stem cells (1) and 2nd generation (2) and 13th generation: 0h ( 1): 71.551±1.380, (2): 72.568±3.0919, 16h(1): 25.600±1.1472, (2): 24.782±2.9133, 28h(1): 3.360±0.5669, 28h(2): 3.439±0.7611. 0h, 16h, 28h: p value = 0.9795, 0.9885, 0.9501.

Figure 6 is a test of wound and repair of bone marrow mesenchymal stem cells between fluorescent pigs and normal pigs: (1) There is no significant difference in the migration and proliferation ability of green fluorescent protein transgenic pigs and (2) normal pig bone marrow mesenchymal stem cells: 0h (1): 74.3464±4.6808, (2): 75.4045±5.8721, (p=0.9838); 16h(1): 30.2290±1.4930, (2): 31.4220±0.9757, (p=0.9885); 28h(1): 2.5177±0.2647, (2): 2.5727±0.3356 (p=0.9719).

Figure 7 is an in vitro differentiation analysis of green fluorescent porcine bone marrow mesenchymal stem cells: (AD) is a hard bone cell differentiation: (A) the leaf stem cells are cultured in a bone differentiation medium for three weeks, and the arrow refers to a precipitate of calcium crystals; The fluorescence of the leaf stem cells differentiated into hard bone cells to express green fluorescent protein; (C) alkaline phosphatase (alkaline phospatase) to detect bone differentiation on the tenth day; (D) ARS staining to detect bone differentiation for the eleventh day Deposition of bone calcium ions. (E, F) for chondrocyte differentiation: (E) Shooting of this mesenchymal stem cell differentiated into chondrocytes expressing green fluorescent protein under fluorescence; (F) Toluidine blue staining detection of cartilage differentiation tissue section micromass pellet showed glycosaminoglycan expression. (GI) is adipocyte differentiation: (G) this leaf stem cell cultured in adipocyte differentiation medium for three weeks with oil droplet formation; (H) shooting under fluorescent light, the mesenchymal stem cells differentiate into adipocytes to express green fluorescent protein (I) Oil Red O detects lipid particles in cells. (J, K, L) Flow cytometry showed that the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes and adipocytes still showed a large amount of green fluorescent protein.

Figure 8 is an in vitro differentiation analysis of fluorescent porcine bone marrow mesenchymal stem cells: (A) is a red fluorescent porcine mesenchymal stem cell; (B) is a red fluorescent protein expressed by a fluorescent microscope; (C) HE staining revealed that the leaf stem cells could differentiate into chondrocytes; (D) Toluidine blue staining to detect cartilage differentiation tissue sections micromass pellet showed glycosaminoglycan expression; (E) for this mesenchymal stem cell culture in adipocyte differentiation medium for three weeks There is oil droplet formation; (F) is the red fluorescent protein expressed by the differentiation of the mesenchymal stem cells into adipocytes under fluorescence; (G) is the detection of lipid particles in this fat cell by Oil Red O; (H) for this mesenchymal The stem cells were cultured in bone differentiation medium for three weeks with precipitation of calcium crystals; (I) for the differentiation of the mesenchymal stem cells into hard bone cells under fluorescence to show red fluorescence; (J) for ARS staining to detect bone differentiation. Deposition of calcium ions in the celestial bone.

Fig. 9 shows in vitro differentiation of fluorescent porcine bone marrow mesenchymal stem cell-like nerve cells: (A) the mesenchymal stem cells are cultured in a neuroblast-like differentiation medium for three weeks; (B) the differentiated neuron-like cells continuously exhibit green fluorescing Photoprotein; (C) is the expression of beta 3 tubulin in such nerve cells by cellular immunostaining.

Figure 10 shows that green fluorescent porcine bone marrow mesenchymal stem cells (GFP-pMSCs) are transdifferentiated into hepatocytes in vitro: (A) observed under a fluorescent microscope, green fluorescent protein is still differentiated after GFP-pMSCs Highly expressed, (B) is the nuclear position determined by DAPI staining, (C) is the expression of albumin (albumin) after differentiation of GFP-pMSCs via red fluorescent secondary antibody and one of goat anti-porcine albumin and antibody (D) ) is a fusion map of the three graphs of (A)(B)(C).

Figure 11 shows the pattern of transdifferentiation of green fluorescent porcine mesenchymal stem cells into insulin-secreting cells induced by cell pellets on day 1 (A), day 1 (B), day 7 (C). The change on day 14 and the persistence of green fluorescent protein during its differentiation (D, E, F) (100X).

Figure 12 is a diagram showing the expression of insulin protein (islet-like cell mass) (200X) in green fluorescent porcine mesenchymal stem cells after differentiation (14 days) by cell immunostaining: (A) observation of green fluorescein under a fluorescent microscope The light pig porcine mesenchymal stem cells differentiate into insulin-like cells and continue to express green fluorescent protein; (B) DAPI stained nucleus; (C) is the red-fluorescent secondary antibody against hamster immunoglobulin Primary antibody, which shows insulin protein detected by differentiated insulin cells; (D) is a fusion map of (A) (B) (C) three maps.

Figure 13 shows the expression of transcription factors and genes of pancreatic islet cells associated with green fluorescent porcine bone marrow mesenchymal stem cells before (A) and after differentiation (B): (A) green fluorescent porcine mesenchymal stem cells before differentiation ( B) Expression of islet cell-associated transcription factors and genes in differentiated green fluorescent porcine bone marrow mesenchymal stem cells. Where: Lane 1: Marker; Lane 2: Blank; Lane 3: Insulin; Lane 4: Glucagon; Lane 5: Glucokinase; Lane 6: Somatostatin; Lane 7: Pax6; Lane 8: Nkx 6.1; Lane 9: Glut 2.

Figure 14 is a graph showing the effect of transplanting green fluorescent porcine islet cells (6 × 10 ⁶ ) derived from green fluorescent porcine bone marrow mesenchymal stem cells on blood glucose of diabetic mice. The hyperglycemia in NOD mice was significantly higher than that in other groups (injection of PBS, EGFP-MSCs, control group) after transplantation of green fluorescent porcine islet cells.

Figure 15 is the survival rate of NOD diabetic mice after transplantation: green fluorescent porcine islet cells (6 × 10 ⁶ ) derived from fluorescent porcine mesenchymal stem cells can survive for at least 20 days and the survival rate (100%), while other groups live up to only 18 days.

Figure 16 is a diagram showing the treatment of fluorescent porcine bone marrow mesenchymal stem cells in bone marrow of osteoporosis mice after 2 months. The photograph was taken under (A) white light, (C) fluorescent and (B) 2 combined (100x). (D) shows the use of green fluorescent protein antibodies in histochemical immunostaining to determine the presence of mesenchymal stem cells of green fluorescent protein transgenic pigs in the bone marrow.

Figure 17 shows the treatment of fluorescent porcine bone marrow mesenchymal stem cells in osteoporosis mice 2 months later, by biochemical immunoassay to detect the presence of bone marrow mesenchymal stem cells in green fluorescent protein transgenic pigs in bone tissue, using green fluorescence Protein antibodies were found on trabecular bone (A) and cortical bone (C) (100x), and bone marrow mesenchymal stem cells of untreated green fluorescent protein transgenic pigs were used as control group (B, D). Magnification map (200x).

Claims (8)

A porcine bone marrow mesenchymal stem cell capable of expressing fluorescent protein, which is a fluorescent reporter gene embedded in the chromosome of the prokaryotic nucleus of an early pig embryo, and is subjected to embryo displacement to produce a gene-transgenic pig that expresses the fluorescent protein systemically, and then From the bone marrow of the obtained fluorescent protein gene-transferred pig fluorescent pig, the adult stem cells with consistent and continuous expression of fluorescent protein are isolated, and further purified to obtain mesenchymal stem cells, wherein the porcine bone marrow mesenchymal stem cells are induced Differentiation produces specific outer, middle, and endoderm cells. The porcine bone marrow mesenchymal stem cells according to claim 1, wherein the fluorescent protein is a green fluorescent protein. The porcine bone marrow mesenchymal stem cells according to claim 1, wherein the porcine bone marrow mesenchymal stem cells are induced to differentiate into skeletal cells, adipocytes, chondrocytes, nerve cells or insulin secreting cells. A method for producing a specific cell expressing a fluorescent protein, comprising: inserting a fluorescent reporter gene into a chromosome of an early porcine embryo pronucleus, and performing embryo displacement to produce a gene-transgenic pig that expresses a fluorescent protein systemically, and then The obtained fluorescent protein gene is transfected into the bone marrow of the pig fluorescent pig, and the adult stem cells which are consistently and continuously exhibiting fluorescent protein are separated, and the mesenchymal stem cells are further purified, and the obtained pig is induced by a specific factor. Bone marrow mesenchymal stem cells are induced to differentiate into specific outer, middle, and endoderm cells that express fluorescent proteins. The method of claim 4, wherein the porcine bone marrow mesenchymal stem cells are induced to differentiate into skeletal cells, adipocytes, chondrocytes, nerve cells or insulin. Secrete cells. A porcine bone marrow mesenchymal stem cell as claimed in claim 1, 2 or 3, which can be used to establish an experimental platform system for studying the source of cell induced differentiation. For example, the porcine bone marrow mesenchymal stem cells of claim 6 can be used to establish an experimental platform system for detecting gene therapy for diabetes. For example, the porcine bone marrow mesenchymal stem cells of claim 6 can be used to establish an experimental platform system for gene therapy detection of osteoporosis.