WO2020242195A1 - Feline wharton's jelly-derived stem cells and preparation method therefor - Google Patents

Abstract

The present invention relates to feline Wharton's jelly-derived stem cells derived from feline Wharton's jelly, and a preparation method therefor. Feline Wharton's jelly-derived stem cells of the present invention maintain a proliferative capacity of up to passages 15 to 20 so as to be easily obtained, and have the capacity to differentiate into bone, cartilage, and fat that is superior to that of stem cells derived from other feline tissue, and thus can be effectively used as a cell therapeutic agent for treating various feline diseases.

Description

Feline Wharton's Jelly-derived Stem Cells and Manufacturing Method thereof

The present invention relates to stem cells derived from feline Wharton jelly and a method for producing the same.

With the recent increase in single-person households, the number of households with companion animals accounts for 25.1% of the total households, and pets being reared are in the order of dogs, cats, and goldfish. With the reinforcement of the family concept of companion animals, the demand for companion animal medicines is also on the rise due to diseases and injuries. In fact, the average sales of veterinary drug manufacturers for the past five years have grown by an annual average of 7.8%. In particular, the growth rate of the companion veterinary drug market is growing at an annual average of 15%, and the share of the entire veterinary drug market is also increasing significantly. Due to the aging phenomenon of companion animals, chronic and degenerative diseases such as diabetes, high blood pressure, and joint disease are also increasing, and the need for appropriate treatment is highlighted. Bone-related diseases that are common in companion animals include hip dysplasia, patella dislocation, shoulder degeneration, elbow arthritis, and leg swelling, and various methods have been attempted to treat them.

Attempts are being made to apply tissue regeneration therapy that has been applied to humans to companion animals recently, and studies are being conducted to treat companion animals using stem cells of companion animals. Stem cells are undifferentiated cells that have the ability to self-replicate and differentiate into two or more different types of cells, and may be classified into embryonic or adult stem cells according to cytological origin. Due to ethical restrictions, embryo-derived stem cells are being studied mainly to use adult stem cells as cell therapeutics, which can be variously extracted from biological bone marrow, brain, liver, pancreas, fat, and cord blood.

In this regard, Korean Patent Literature KR 10-2018-0122818 discloses canine amniotic membrane-derived multipotent stem cells, and studies for treating diseases of animals by isolating/culturing stem cells from adipose tissue of animals are reported. Has been done. However, compared with research reports on stem cell and stem cell therapy in canine animals, many studies on stem cell therapy in feline animals have not yet been reported. However, in stem cell therapy, it is very important to use autologous or allogeneic stem cells to solve the problem of tissue compatibility, and there is an urgent need for research on this.

In order to solve the above problems, the present inventors are conducting research on stem cells that can be used for tissue regeneration treatment of feline animals, and can effectively separate stem cells from feline Wharton jelly, isolated feline Wharton jelly. It was confirmed that the derived stem cells exhibited superior differentiation ability compared to stem cells derived from other tissues, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a composition for regenerating feline tissues including stem cells derived from Feline Wharton Jelly and the same.

Accordingly, an object of the present invention is to provide stem cells derived from cat Wharton jelly, characterized by expressing surface factors positive for CD105, CD90 and CD44 and negative for CD45, CD34 and CD14.

It is also an object of the present invention to provide a composition for regeneration of feline tissues comprising stem cells derived from cat Wharton jelly.

In addition, the object of the present invention is (1) obtaining a Feline-derived Wharton jelly; And (2) extracting and culturing cells from the Wharton jelly. It is to provide a method for producing stem cells derived from Feline Wharton jelly comprising a.

In addition, the object of the present invention is to cultivate stem cells derived from cat Wharton jelly in chondrocyte differentiation medium; It is to provide a method for differentiating stem cells derived from cat Wharton jelly into chondrocytes comprising a.

In addition, the object of the present invention is to cultivate stem cells derived from cat Wharton jelly in adipocyte differentiation medium; It is to provide a method of differentiating stem cells derived from cat Wharton jelly into adipocytes, including.

In addition, the object of the present invention is to cultivate stem cells derived from cat Wharton jelly in a bone cell differentiation medium; It is to provide a method of differentiating stem cells derived from cat Wharton jelly into bone cells comprising a.

In addition, it is an object of the present invention to provide a method for tissue regeneration of a feline animal comprising the step of treating stem cells derived from Feline Wharton jelly on a feline animal.

The feline Wharton jelly-derived stem cells of the present invention maintain proliferative ability from passages 15 to 20, making it easy to obtain, as well as superior differentiation into bone, cartilage, and fat compared to stem cells derived from other feline tissues. It can be effectively used as a cell therapy for treating diseases of the disease.

1 is a diagram showing the Wharton jelly obtained by cesarean section of a cat.

2 is a diagram showing the results of confirming the morphology of stem cells derived from cat Wharton jelly.

3 is a diagram showing the results of confirming stem cell specific markers in cat Wharton jelly-derived cells.

4 is a diagram showing the results of confirming the cumulative population doubling level (CPDL) according to the passage of cells derived from cat Wharton jelly.

5 is a diagram showing the results of confirming the expression of surface antigens of stem cells derived from cat Wharton's jelly through FACS.

6 is a diagram showing the results of confirming the degree of bone differentiation through Alizarin Red S and Von Kossa staining after culturing stem cells derived from cat Wharton's jelly in a bone differentiation specific medium. 6A to E are results after inducing differentiation in an osteodifferentiation medium, and F to J are results of cells in an undifferentiated state. K in Figure 6 is a diagram showing the quantification of the Alizarin Red S staining results (Control: undifferentiated control, Osteogenic: bone differentiation induction experimental group).

7 is a diagram showing the result of confirming the expression pattern of MSX2, a bone-related specific gene, in stem cells derived from cat Wharton's jelly by qRT-PCR technique.

8 is a diagram showing the results of confirming the degree of fat differentiation through Oil Red O staining after culturing stem cells derived from cat Wharton's jelly in a fat differentiation specific medium. 8A to 8C are results after inducing differentiation in an adipogenic differentiation medium, and D to F are results of cells in an undifferentiated state. 8G is a diagram showing the quantification of Oil Red O staining results (Control: undifferentiated control, Adipogenic: fat differentiation inducing experimental group).

9 is a diagram showing the results of confirming the expression patterns of fat-related specific genes LPL, LEPTIN and FABP4 in stem cells derived from cat Wharton's jelly by qRT-PCR technique.

FIG. 10 is a diagram showing the result of confirming the degree of differentiation through the formation of cartilage pellets and toluidine blue staining, which is a cartilage-specific staining, after culturing stem cells derived from cat Wharton's jelly in a cartilage differentiation specific medium. 10A is a diagram showing the result of confirming that a pellet in the form of cartilage was formed. B of FIG. 10 is a diagram showing the results confirming that cartilage differentiation was induced through toluidine blue staining.

FIG. 11 is a diagram showing the result of confirming the expression pattern of COL2A1, a specific gene related to cartilage, by qRT-PCR technique after inducing differentiation in a cartilage differentiation medium.

12 is a diagram showing the result of confirming the morphological characteristics of stem cells after extracting stem cells from cat bone marrow and fat.

13 is a diagram showing the results of comparing the expression of the bone differentiation-specific marker Sparc over 1 to 3 weeks after inducing bone differentiation from stem cells isolated from cat Wharton's jelly (WJ), bone marrow (BM) and fat (AD) to be.

14 is a diagram showing the results of comparing the expression of bone differentiation-specific marker Msx2 over 1 to 3 weeks after inducing bone differentiation from stem cells isolated from cat Wharton's jelly (WJ), bone marrow (BM) and fat (AD). to be.

Figure 15 is a view showing the results of comparing the expression of the bone differentiation specific marker Col1a1 over 1 to 3 weeks after inducing bone differentiation from stem cells isolated from cat Wharton's jelly (WJ), bone marrow (BM) and fat (AD) to be.

FIG. 16 is a staining result obtained by inducing bone differentiation from stem cells isolated from cat Wharton's jelly (WJ), bone marrow (BM) and fat (AD) and performing bone specific staining (alizarin red S) over 1 to 3 weeks (A ) And the result of quantification thereof (B).

The present invention relates to stem cells derived from Feline Wharton Jelly, characterized by expressing surface factors that are positive for CD105, CD90 and CD44 and negative for CD45, CD34 and CD14.

The stem cells derived from Feline Wharton Jelly of the present invention are extracted, separated, and obtained from feline animals, have excellent tissue compatibility when transplanted into feline animals, can lower the possibility of tumor occurrence, and are obtained from Wharton Jelly, so that smooth supply and demand is possible. In addition, the stem cells derived from Feline Wharton jelly of the present invention may be characterized by superior differentiation ability into bone, cartilage, or fat cells compared to stem cells isolated from other tissues.

In the present invention, the feline is a member of the Felidae family (ie, Felid). The feline animal may include a feline family (felinae) or a pantherinae family (pantherinae), and the feline family animal includes the genus Feline, the genus Puma, the genus cheetah, the genus Holly, and the genus Felis Catus is a domestic cat. (Felis catus) and Felis silvestris catus.

In the present invention, Wharton's Jelly can be used interchangeably with Barton's Jelly, and can be obtained from the umbilical cord of a feline animal. Wharton jelly obtained from the umbilical cord of a feline animal can be obtained through a mechanical and physical treatment process of separating the umbilical cord from a pregnant cat and removing the umbilical cord membrane and blood vessels using a scalpel and tweezers. In order to extract and separate stem cells from the obtained Wharton jelly, a physical and chemical treatment process may be applied to the Wharton jelly. Specifically, the blood vessel was separated using forceps and the tissue was cut finely, followed by enzymatic treatment such as collagenase. Can handle type I.

The stem cell derived from Feline Wharton jelly of the present invention exhibits a characteristic that the proliferation rate does not decrease even when the passage increases, and the proliferation ability is maintained even at passages 10 to 20, preferably passages 15 to 20, and more preferably passages 15 to 18. It can be a cell. In particular, the stem cells of the present invention maintain the proliferative ability in an undifferentiated state, and undifferentiated refers to a state in which differentiation does not occur and yet exhibits characteristics as stem cells.

In addition, the stem cells derived from Feline Wharton jelly of the present invention may be cells capable of differentiating bone, fat or cartilage, and by culturing the obtained stem cells in an appropriate medium for inducing bone, fat or cartilage differentiation, Bone, fat, and chondrocytes can be obtained.

In the stem cells derived from Feline Wharton's jelly of the present invention, the expression of MSX2, which is a bone-related specific gene, is increased during bone cell differentiation, and the expression of LPL, LEPTIN, and FABP4, which are adipose-related specific genes, is increased during differentiation of adipocytes, and cartilage during differentiation of chondrocytes. These stem cells show an increase in the expression of the related specific gene COL2A1.

As a medium for inducing bone differentiation in the present invention, a medium known in the art optimized for bone differentiation may be used without limitation, and dexamethasone, ascorbic acid, β-glycrophosphate, and ascorbic acid A medium containing -2-phosphate (ascorbic acid-2-phosphate) may be used. For example, in one embodiment of the present invention, StemPro Osteogenesis Differentiation Kit (Gibco, USA) was used.

In the present invention, as a medium for inducing adipogenic differentiation, a medium known in the art optimized for adipogenic differentiation may be used without limitation, and dexamethasone, indomethacin, insulin and IBMX (3 -isobutyl-1-methylxanthine) can be cultured in a medium containing adipocytes to induce differentiation into adipocytes. For example, in one embodiment of the present invention, StemPro Adipogenesis Differentiation Kit (Gibco, USA) was used.

In the present invention, as a medium for inducing cartilage differentiation, a medium known in the art optimized for cartilage differentiation may be used without limitation, such as dexamethasone, acetylsalicylic acid, sodium pyruvate, proline, ITS, and growth factors. A differentiation medium containing may be used, for example, in one embodiment of the present invention, StemPro Chondrogenesis Differentiation Kit (Gibco, USA) was used.

In addition, the present invention provides a composition for regeneration of feline tissues comprising stem cells derived from feline Wharton jelly.

In addition, the present invention provides a method for tissue regeneration of a feline animal comprising the step of treating stem cells derived from feline Wharton jelly on a feline animal.

The composition for regeneration of feline tissues of the present invention comprises a cell therapy for tissue regeneration, and contains stem cells derived from Feline Wharton's jelly having the ability to differentiate into cartilage, bone, and fat as an active ingredient, thereby transplanting them into cats suffering from diseases. It is characterized in that it can effectively induce regeneration of damaged tissues of cats without rejection to tissue bonding.

Cell therapy is a drug that is used for treatment, diagnosis, and prevention purposes with cells and tissues manufactured through isolation, culture and special manipulation (US FDA regulations), and is a living autologous, allogeneic, or It refers to a pharmaceutical product in which these cells are used for the purpose of treating, diagnosing, and preventing diseases through a series of actions such as proliferation and selection of heterogeneous cells in vitro or changing the biological characteristics of cells by other methods. Preferably, the composition for tissue regeneration of the present invention can be used for treatment of cartilage damage, cartilage defects, bone defects, tendon-ligament defects, adipose tissue defects, etc. of feline animals, and stem cells derived from feline Wharton jelly are used in feline animals. By treatment, it can be used for tissue regeneration of feline animals, and preferably, it can be used for treatment of cartilage damage, cartilage defect, bone defect, tendon-ligament defect, adipose tissue defect, and the like of feline animals.

In the present invention, the term "cartilage defect" refers to a case where there is a damage, defect, or lack of cartilage contained in the body, for example, cartilage trauma, cartilage rupture, cartilage softening, cartilage necrosis, osteochondritis, cartilage Including, but not limited to, defects or osteoarthritis.

In addition, the stem cells derived from Feline Wharton's jelly of the present invention can be used for the purpose of treating lesions of articular cartilage by administering them into the joints of a feline animal, or treating or preventing them by administering them to tendons or ligaments. For example, by administering the stem cells derived from Feline Wharton jelly of the present invention to a joint, tendon, or ligament site, recovery or adjustment to the damaged area of the tissue is achieved, or stem cells derived from Feline Wharton jelly of the present invention It can be used to reorganize tissues of joints (eg, knee joints, etc.) using stem cell-derived materials such as cartilage tissue constructs or to treat them by methods such as regeneration.

The dosage of the composition for tissue regeneration of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration may be administered once a day, or may be divided several times, and the dosage does not limit the scope of the present invention in any way.

The composition for tissue regeneration may additionally include a pharmaceutically acceptable carrier known in the art for the purpose of inducing tissue regeneration in feline. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, a lubricant, and a preservative. The pharmaceutical composition of the present invention can be prepared in the form of various formulations according to commonly used techniques.

Feline Wharton jelly-derived stem cells of the present invention include the steps of: (1) obtaining Wharton jelly derived from feline; And (2) extracting and culturing cells from the Wharton jelly. It can be manufactured through. Therefore, the present invention (1) to obtain a Wharton jelly derived from feline; And (2) extracting and culturing cells from the Wharton jelly. It provides a method for producing stem cells derived from Feline Wharton jelly comprising a.

The step (1) is a process of obtaining Wharton's jelly from a pregnant feline animal, and can be obtained by separating the umbilical cord and removing the umbilical cord membrane and blood vessels from the umbilical cord through a physical process.

The step (2) is a step of extracting and culturing cells from the obtained Wharton jelly, where "extraction" of the cells may be performed through a physical or chemical treatment process. Specifically, the cell extraction is performed by separating blood vessels from Wharton's jelly and cutting the tissue finely; And it may be carried out through the step of enzymatic treatment on the cut tissue, the enzyme may be a collagenase degrading enzyme, more preferably a collagenase type I. The collagenase-degrading enzyme may be treated at a concentration of 0.1mg/ml to 5mg/ml, preferably 0.5mg/ml to 3mg/ml, more preferably 1mg/ml to 2mg/ml.

In the step (2), the "culture" is a step of culturing the cells separated by enzyme treatment in a culture medium to separate and proliferate, and a cell culture medium known in the art can be used without limitation, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM-F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), LG-MEM, IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete Medium, AminoMaxII complete Medium, Chang's Medium, and MesenCult-XF Medium. For the crab, a medium containing low-concentration glucose can be used. When a medium containing low-concentration glucose is used, an LG-DMEM medium may be preferably used, and FBS may be added to the medium, and FBS may be added at a concentration of 5 to 15% (v/v). The low-concentration glucose DMEM medium may contain 0.1 g/L to 3 g/L, more preferably 0.5 to 1.5 g/L of glucose, but is not limited thereto.

The stem cells derived from Feline Wharton jelly of the present invention are cells having differentiation ability into cartilage, fat or bone, and culturing them in a chondrocyte differentiation medium, an adipocyte differentiation medium, or a bone cell medium; It is possible to provide a method for differentiating stem cells derived from cat Wharton's jelly into chondrocytes, adipocytes, or bone cells.

Hereinafter, the present invention will be described in detail by examples and experimental examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Isolation and culture of cat Wharton jelly-derived cells

Wharton's jelly was obtained from a pregnant cat (4~5kg) through aseptic procedure (Cesarean section). Propofol (5mg/kg, myeongmun pharmaceutical, Korea) was administered to pregnant cats to induce anesthesia, and anesthesia was maintained using a respiratory anesthetic (Isoflurane). The skin was prepared aseptically, and cesarean section was performed through incision of the lower abdominal skin, subcutaneous and ringworm, and Wharton jelly was obtained in a sterile state, and the obtained Wharton jelly is shown in FIG. In order to aseptically extract and culture stem cells from the extracted Wharton jelly, Wharton jelly was washed 2-3 times with 0.9% PBS to remove blood and cell debris, and blood vessels were physically removed using forceps. The treated Wharton jelly was chopped using a surgical knife, and placed in a 2mg/ml collagenase type I solution at 37°C for 3 hours. Thereafter, centrifugation was performed at 3000 rpm for 5 minutes to collect the cell pellet, and culture was performed under aliquoting in a cell culture dish. For cell culture, a medium containing 10% FBS (Fetal bovine serum; Gibco, USA) added to a low-concentration glucose DMEM medium (LG-DMEM; Gibco, USA) was used, and the extracted cells were cultured in a 5% CO ₂ incubator and the medium was It was replaced 3 times a week. The extracted cells were set to passage 0, and then the cells were maintained through passage culture.

Example 2. Confirmation of stem cell ability of cat Wharton jelly-derived cells

In order to confirm whether the cells obtained through the method of Example 1 were stem cells derived from cat Wharton's jelly, the morphology of stem cells, stem cell specific markers, growth ability, and surface antigen expression were confirmed.

Confirmation of stem cell morphology

Adult mesenchymal stem cells grow attached to the bottom of a plastic culture dish and are characterized by a thin and elongated shape. This was compared with the morphology of the cells present in the medium of the present invention, and the results are shown in FIG. 2.

As shown in Figure 2, it was confirmed that the cells isolated from the cat Wharton's jelly exhibit the same morphology as the adult mesenchymal stem cells.

Stem cell specific marker identification

In order to confirm that the extracted cells are actual stem cells, the expression patterns of the stem cell-specific markers SOX2, KLF4, and C-MYC were confirmed through RT-PCR, and the results are shown in FIG. 3. Gene expression analysis was performed by the following method through RT-PCR technique. Total RNA was extracted from the cultured cells using RNeasy minikit (Qiagen, Germany). RNA concentration was measured by Nanodrop 2000 (ThermoScientific, USA). CDNA was prepared with 1 mg of total RNA for reverse transcription using Superscript II reverse transcriptase (Invitrogen, USA) and oligo dT primer (Invitrogen, USA). The cDNA was amplified using a T100™ Thermal Cycler (Biorad, USA) and the PCR product was visualized using a 3% agarose gel.

As shown in Figure 3, cells derived from the cat Wharton jelly showed the expression of stem cell-specific markers SOX2, KLF4, C-MYC, through which it was confirmed that the cells are stem cells.

Confirmation of stem cell growth characteristics

CPDL (cumulative population doubling level) was measured to confirm whether the cells isolated in Example 1 exhibit the growth ability of stem cells. The proliferation and growth efficiency of the isolated stem cells was determined by the accumulated individual doubling level (CPDL) using the formula CPDL = ln (Nf / Ni) ln2. Where Ni is the number of initial cultured cells, Nf is the number of final harvested cells, and ln is the natural logarithm. The isolated stem cells (5×10 ⁴ cells) were plated 3 times in a 6-well culture plate (Corning, USA) and then subcultured 4 days later. The final number of cells was counted and 5 × 10 ⁴ cells were repeatedly subcultured to measure cell growth capacity, and the results are shown in FIG. 4.

As shown in FIG. 4, stem cells derived from cat Wharton's jelly exhibited the characteristic of increasing CPDL as passage was increased, and the proliferation rate did not decrease until passage 16 and the growth ability was continuously maintained. These results show that the stem cells derived from cat Wharton's jelly isolated in Example 1 have the ability to self-proliferate and regenerate stem cells, and can exhibit the same growth characteristics of adult stem cells.

Stem cell specific surface antigen expression confirmation

Stem cells express specific surface antigens of stem cells, and FACS (fluorescence-activated cell sorter) analysis was performed to confirm this, and the results are shown in FIG. 5.

As shown in FIG. 5, stem cells derived from cat Wharton's jelly showed positive expression for human markers CD105, CD90 and CD44, and negative expression for CD45, 34 and 14.

By synthesizing all of the above characteristics, it was confirmed that the cells extracted and isolated through Example 1 are stem cells, and stem cells derived from cat Wharton jelly showing all of the morphological characteristics, proliferation ability, and surface marker expression characteristics of the stem cells were obtained. I did.

Example 3. Confirmation of bone differentiation ability of stem cells derived from cat Wharton jelly

It was confirmed whether the stem cells derived from cat Wharton's jelly extracted in Example 1 had bone differentiation ability. The stem cells derived from cat Wharton's jelly obtained in Example 1 were placed in a bone differentiation specific medium (StemPro Osteogenesis Differentiation Kit; Gibco, USA), cultured for bone differentiation was performed for 3 weeks, and the degree of bone differentiation with undifferentiated cells was compared. After 3 weeks, bone-specific staining of Alizarin Red S and Von Kossa was performed to confirm the degree of bone differentiation, and the results of Alizarin Red S staining were quantified through absorbance analysis, and the results are shown in FIG. 6.

As shown in Figure 6, unlike the undifferentiated experimental group F to J of Figure 6, in all of A to E of Figure 6 that induced bone differentiation, red bone differentiation was confirmed, and as a result of quantification, a remarkable increase in absorbance was confirmed. It was confirmed that the jelly-derived stem cells have excellent bone differentiation ability.

The expression pattern of MSX2, a bone-related specific gene, was additionally confirmed using the qRT-PCR technique to confirm the ability of bone differentiation versus undifferentiation, and the results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that bone differentiation was effectively induced by increasing the expression of MSX2, a bone-related specific gene, in stem cells derived from cat Wharton's jelly by 1.2 times.

Example 4. Confirmation of fat differentiation ability of stem cells derived from cat Wharton jelly

It was confirmed whether the stem cells derived from cat Wharton's jelly extracted in Example 1 had the ability to differentiate into fat. The stem cells derived from cat Wharton jelly obtained in Example 1 were placed in an adipogenic differentiation specific medium (StemPro Adipogenesis Differentiation Kit; Gibco, USA), cultured for adipogenic differentiation was performed for 3 weeks, and the degree of adipogenic differentiation with undifferentiated cells was compared. After 3 weeks, in order to check the degree of fat differentiation, oil red O staining was performed, which is a fat-specific staining, and the result of quantification through absorbance analysis is shown in FIG. 8.

As shown in FIG. 8, compared to the experimental groups (E, F) in which undifferentiated cells were stained, red stained portions of cells cultured in the adipocyte differentiation medium were confirmed (A), fat cells (fatty droplet) was confirmed (B). As shown in G of FIG. 8, the Oil Red O staining result was quantified using absorbance and compared with undifferentiated to confirm the degree of differentiation, it was confirmed that about 5 times of differentiation was achieved compared to the undifferentiated control group. These results show that stem cells derived from cat Wharton's jelly can effectively differentiate into adipocytes.

Additionally, the expression patterns of fat-related specific genes LPL, LEPTIN, and FABP4 were compared in undifferentiated vs. adipocytes using the qRT-PCR technique, and qRT-PCR was performed in the following manner. Total RNA was extracted from the cultured cells using RNeasy minikit (Qiagen, Germany). RNA concentration was measured by Nanodrop 2000 (ThermoScientific, USA). CDNA was prepared with 1 mg of total RNA for reverse transcription using Superscript II reverse transcriptase (Invitrogen, USA) and oligo dT primer (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) was performed by mixing cDNA with primers and LightCycler®480 SYBR Green I Master (Roche Diagnostics, Germany). qRT-PCR was performed using LightCycler 480 II equipped with software (Roche applied science, Germany) provided according to the manufacturer's instructions, and RNA expression levels were compared after standardization for endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results of confirming the expression patterns of LPL, LEPTIN and FABP4 are shown in FIG. 9.

As shown in FIG. 9, the expression of LPL and LEPTIN was increased by about 2 times or more in the stem cell group derived from the cat Wharton jelly of the present invention compared to the control group, and FABP4 showed a very remarkable increase in expression.

Example 5. Confirmation of cartilage differentiation ability of stem cells derived from cat Wharton jelly

It was confirmed whether the stem cells derived from cat Wharton's jelly extracted in Example 1 had the ability to differentiate into cartilage. The stem cells derived from cat Wharton's jelly obtained in Example 1 were attached to the bottom of a 15 ml polypropylene tube to apply a cartilage differentiation specific medium (StemPro Chondrogenesis Differentiation Kit; Gibco, USA), and culture for cartilage differentiation was performed for 4 weeks and undifferentiated cells And the degree of cartilage differentiation were compared. The degree of cartilage differentiation was confirmed by confirming the formation of pellets in the form of cartilage and by dyeing toluidine blue, which is a cartilage-specific dye, and the expression pattern of the cartilage-related specific gene COL2A1 was confirmed using the same qRT-PCR method as in Example 4. Is shown in FIGS. 10 and 11.

As shown in FIG. 10, it was confirmed that stem cells derived from cat Wharton's jelly generate a pellet form (A), and they were stained with toluidine blue (B). In addition, as shown in FIG. 11, the expression of the cartilage-related specific gene COL2A1 was increased by about 7 times or more compared to the control group, confirming that the chondrocyte differentiation ability of the cat Wharton jelly-derived stem cells was very excellent.

Comparative example. Extraction and Characterization of Cat-derived Stem Cells by Tissue

In order to confirm whether adult stem cells can be effectively obtained from cat tissues other than the cat Wharton jelly-derived stem cells of Example 1, stem cells were extracted from feline bone marrow and fat. Cat bone marrow-derived stem cells used in the experiment were obtained through bone marrow puncture. Anesthesia was induced by administration of propofol (5mg/kg, myeongmun pharmaceutical, Korea) to cats, and anesthesia was maintained using a respiratory anesthetic (Isoflurane). Femurs and buttocks of cats were aseptically prepared, 0.5 cm of skin was incised, a needle for bone marrow puncture was inserted through the cortex through the cortex of the femur on the body side, and bone marrow was aseptically collected using a 10 ml syringe. The collected solution was removed from cell debris and bone tissue using a cell strainer (100um pore size, Falcon, USA), and then centrifuged at 3000rpm for 20 minutes to collect cell pellets and dispense them into a cell culture dish. Culture was carried out. For cell culture, a medium containing 10% FBS (Fetal bovine serum; Gibco, USA) added to a low-concentration glucose DMEM medium (LG-DMEM; Gibco, USA) was used, and the extracted cells were cultured in a 5% CO ₂ incubator and the medium was It was replaced 3 times a week. The extracted cells were set to passage 0, and then the cells were maintained through passage culture.

Stem cells derived from cat fat used in the experiment were collected from adipose tissue. The anesthesia protocol is as follows. Propofol (5mg/kg, myeongmun pharmaceutical, Korea) was administered to induce anesthesia, and anesthesia was maintained using a respiratory anesthetic (Isoflurane). The cat's abdomen was prepared aseptically, and only subcutaneous adipose tissue was aseptically collected from the third point of the lower abdomen. Thereafter, in order to extract and culture stem cells, the adipose tissue was washed 2-3 times with 0.9% PBS to remove blood and cell debris, and the surrounding blood vessels were physically removed using forceps. The treated adipose tissue was chopped using a surgical knife, and placed in a 2mg/ml collagenase type I solution at 37°C for 1 hour. Thereafter, centrifugation was performed at 3000 rpm for 5 minutes to collect the cell pellet, and the cell pellet was dispensed into a cell culture dish and cultured. For cell culture, a medium containing 10% FBS (Fetal bovine serum; Gibco, USA) added to a low-concentration glucose DMEM medium (LG-DMEM; Gibco, USA) was used, and the extracted cells were cultured in a 5% CO ₂ incubator and the medium was It was replaced 3 times a week. The extracted cells were set to passage 0, and then the cells were maintained through passage culture.

Fig. 12 shows the results of confirming the morphological characteristics of the obtained feline bone marrow derived mesenchymal stem cells (feline BM-MSCs) and adipose derived stem cells (feline adipose tissue derived mesenchymal stem cells; feline AD-MSCs). It is shown in A and B of.

12A and 12B, both feline bone marrow-derived adult stem cells and feline fat-derived adult stem cells showed morphological characteristics of stem cells.

Comparative Example 1.1. Bone specific marker expression comparison

In order to compare the osteodifferentiation ability of cat Wharton's jelly-derived stem cells and bone marrow and adipose-derived stem cells, the expression of bone-specific markers Sparc, Msx2 and Col1a1 was confirmed in these. Specifically, all the extracted cells were induced to differentiate for 3 weeks using a bone differentiation specific medium (StemPro Osteogenesis Differentiation Kit; Gibco, USA) in the same manner as in Example 3, and qRT-PCR for the expression patterns of Sparc, Msx2 and Col1a1 Through comparison and analysis, the results are shown in FIGS. 13 to 15.

As shown in FIG. 13, stem cells derived from cat Wharton jelly showed the highest Sparc expression over 1 to 3 weeks, and in particular, about 3 weeks after induction of bone differentiation, about 7 compared to bone marrow and adipose-derived stem cells. It was confirmed that the differentiation ability was significantly higher than that of twice.

As shown in FIG. 14, stem cells derived from cat Wharton's jelly showed similar or somewhat lower differentiation ability to those of bone marrow-derived stem cells in the first week of induction of bone differentiation, but the bone marrow-derived stem cells decreased the expression of bone-specific markers in the third week. Unlike that, it was confirmed that the expression of the marker increased in proportion to the induction time of bone differentiation. In addition, compared to adipose-derived stem cells, it showed remarkably excellent marker expression.

As shown in FIG. 15, the expression of the bone-specific marker Col1a1 was continuously increased over the 1st to 3rd weeks of the induction of bone differentiation in the cat Wharton's jelly-derived stem cells, and at the 3rd week, significantly superior marker expression compared to the bone marrow and adipose-derived stem cells. Shown.

These results show the excellent osteodifferentiation effect of cat Wharton jelly-derived stem cells.

Comparative Example 1.2. Comparison of bone differentiation ability through bone specific staining

In order to compare the osteodifferentiation ability of cat Wharton's jelly-derived stem cells and bone marrow and adipose-derived stem cells, the extracted cells were all in the same manner as in Example 3 using a bone differentiation specific medium (StemPro Osteogenesis Differentiation Kit; Gibco, USA) 3 Bone-specific staining (alizarin red S) was performed at week 1, 2, and 3 weeks after induction of bone differentiation in undifferentiated state and bone differentiation was performed, and the degree of staining was checked, and the staining degree was quantitatively compared.

As shown in FIG. 16, at 3 weeks after induction of bone differentiation, the degree of bone-specific staining (alizarin red S) of Wharton's jelly-derived stem cells was the highest in the naked eye compared to stem cells derived from other tissues (A), and the result of quantifying these 3 Compared to bone marrow and adipose-derived stem cells that do not show significant differences from undifferentiated over weeks, the Wharton's jelly-derived stem cells of the present invention showed a very remarkable increase in staining according to the induction time of bone differentiation, and based on these results, the bone differentiation ability was different. It was confirmed to be significantly superior to stem cells derived from cat tissue.

Claims (12)

Stem cells derived from Feline Wharton Jelly, characterized by expressing surface factors positive for CD105, CD90, and CD44 and negative for CD45, CD34 and CD14. The stem cells of claim 1, wherein the feline Wharton jelly-derived stem cells maintain proliferative capacity from passages 15 to 20. The stem cells of claim 1, wherein the feline Wharton jelly-derived stem cells have bone, fat, or cartilage differentiation ability. The method of claim 1, wherein the feline Wharton jelly-derived stem cells are (1) obtaining Feline-derived Wharton jelly; And (2) extracting and culturing cells from the Wharton jelly; Stem cells derived from Feline Wharton Jelly, characterized in that produced through. A composition for regeneration of feline tissue comprising stem cells derived from feline Wharton jelly according to any one of claims 1 to 4. The composition for regeneration of feline tissue according to claim 5, wherein the tissue is bone, cartilage or fat of a feline animal. (1) obtaining Feline-derived Wharton jelly; And (2) extracting and culturing cells from the Wharton jelly; Method for producing stem cells derived from Feline Wharton jelly comprising a. The method of claim 7, wherein the culture is performed in a low-concentration glucose DMEM (Dulbecco's Modified Eagle's Medium-low glucose) medium. Culturing the stem cells derived from Feline Wharton jelly of claim 1 in a chondrocyte differentiation medium; Method for differentiating the stem cells derived from Feline Wharton jelly into chondrocytes comprising a. Culturing the stem cells derived from Feline Wharton jelly of claim 1 in an adipocyte differentiation medium; Method for differentiating the stem cells derived from Feline Wharton jelly into adipocytes comprising a. Culturing the stem cells derived from Feline Wharton jelly of claim 1 in a bone cell differentiation medium; Method for differentiating stem cells derived from Feline Wharton jelly into bone cells comprising a. Feline Wharton jelly-derived stem cells are treated in a feline animal; feline tissue regeneration method comprising a.

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