KR20130019459A - Process for expanding amniotic fluid-derived mesenchymal stem cells and process for preparing grafting constructs comprising the same - Google Patents

Abstract

PURPOSE: A method for proliferation of amniotic fluid-derived mesenchymal stem cells and a method for preparing a structure containing the stem cells for transplantation are provided to effectively differentiate the stem cells into chondrocytes in vivo. CONSTITUTION: A method for proliferation of amniotic fluid-derived mesenchymal stem cells comprises a step of culturing the stem cells in a medium containing fibroblast growth factor-2(FGF-2), epidermal growth factor(EGF), or fibroblast growth factor-2(FGF-2) and epidermal growth factor(EGF). A method for preparing a structure for transplantation to generate cartilage in vivo comprises: a step of proliferation of the stem cells; and a step of encapsulating the stem cells in a fibrin hydrogel containing transforming growth factor(TGF)-beta3. [Reference numerals] (AA) Proliferation-C; (BB) Proliferation-B; (CC) Proliferation-A; (DD) Proliferation-D

Description

Process for expanding amniotic fluid-derived mesenchymal stem cells and process for preparing grafting constructs comprising the same

The present invention relates to a method of proliferating amniotic fluid-derived mesenchymal stem cells and a method for producing a transplant construct comprising the same, more specifically fibroblast growth factor-2 (FGF-2) and / Or a method of proliferating amniotic fluid-derived mesenchymal stem cells, comprising culturing amniotic fluid-derived mesenchymal stem cells in a growth medium containing epidermal growth factor (EGF); And it relates to a method for producing a transplant structure for cartilage production in vivo comprising the proliferated amniotic fluid-derived mesenchymal stem cells.

In recent decades, stem cells have emerged as potential cell sources based on cell therapies for tissue regeneration. However, it is not easy to regulate stem cells to achieve differentiation into the desired cell morphology in vitro and in vivo. Many studies therefore focus on differentiating stem cells into specific cells using specific types of drugs and growth factors.

Adult stem cells derived from bone marrow, adipose tissue, and placenta have been studied as an important cell source of tissue regeneration through cartilage differentiation and formation of damaged bone tissue. Mesenchymal stem cells (MSCs) derived from several types of tissues have been shown to have the ability to differentiate into mesodermal cell lines. Therefore, MSCs have been proposed as complementary raw materials for tissue regeneration in the damage area associated with osteoarthritis and osteoporosis.

In order to apply mesenchymal stem cells of various origin to cell therapy, it is required to proliferate each mesenchymal stem cell effectively for a long time without changing the chromosome while maintaining stem cellity. In addition, since mesenchymal stem cells exhibit different characteristics according to their origin (for example, bone marrow, adipose tissue, placenta, etc.), each mesenchymal stem cell applied to cell therapy establishes a proliferation method suitable for each characteristic. It is required to do. To date, adult-derived stem cells, ie, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, and the like have been studied for proliferation and differentiation by various researchers.

Recently, De Coppi et al. Have reported the results of isolating mesenchymal stem cells from amniotic fluid and evaluating the applicability of the amniotic fluid-derived mesenchymal stem cells as cell therapeutics (De Coppi, P., Bartsch, G., Jr., Siddiqui, MM, Xu, T., Santos, CC and Perin, L., et al., Isolation of amniotic stem cell lines with potential for therapy, Nat Biotechnol 25 (1), 2007, 100-106). These amniotic fluid-derived mesenchymal stem cells can be isolated from amniotic fluid obtained through amniocentesis, which is performed on prenatal pregnant women for early diagnosis of hereditary genetic diseases. There is an advantage. However, amniotic fluid-derived mesenchymal stem cells, unlike other mesenchymal stem cells, have not yet developed a stable proliferation method. De Coppi et al. Reported that they can grow by culturing amniotic fluid-derived mesenchymal stem cells using α-MEM medium supplemented with fetal bovine serum, glutamine, Chang B (Irvine Scientific), Chang C (Irvine Scientific), and antibiotics. However, this has not been reproduced by other researchers.

The present inventors conducted various studies to develop a method for stably proliferating amniotic fluid-derived mesenchymal stem cells for a long time. In particular, the inventors analyzed the growth efficiency of various growth factors, and as a result, specific growth factors, ie, fibroblast growth factor-2 (FGF-2) and / or epidermal growth factor (epidermal growth factor). It has been found that culturing amniotic fluid-derived mesenchymal stem cells in the presence of a factor (EGF) can stably propagate amniotic fluid-derived mesenchymal stem cells for a long time without chromosomal abnormalities. In addition, it was confirmed that the amniotic fluid-derived mesenchymal stem cells thus proliferated can be effectively differentiated into chondrocytes in vivo.

Accordingly, the present invention comprises culturing amniotic fluid-derived mesenchymal stem cells in the presence of fibroblast growth factor-2 (FGF-2) and / or epidermal growth factor (EGF). It is an object to provide a proliferation method.

In addition, an object of the present invention is to provide a method for producing a transplant construct for cartilage generation in vivo, including the amniotic fluid-derived mesenchymal stem cells proliferated as described above.

According to one aspect of the invention, there is provided either (i) fibroblast growth factor-2 (FGF-2) or (ii) epidermal growth factor (EGF) or (iii) fibroblast growth factor-2 (FGF-2) in basal medium. And culturing amniotic fluid-derived mesenchymal stem cells in a growth medium comprising epidermal growth factor (EGF).

In the growth method according to the present invention, the growth medium is added to (i) fibroblast growth factor-2 or (ii) epidermal growth factor or (iii) fibroblast growth factor-2 and epidermal growth factor to the basal medium; It may also be a medium to which fibroblast growth factor-2, fetal bovine serum, 2-mercaptoethanol, MEM non-essential amino acids, and antibiotics are added.

In one embodiment, the proliferation medium is (i) 1-10 ng / ml fibroblast growth factor-2 or (ii) 1-20 ng / ml epidermal growth factor or (iii) 1-10 ng in basal medium. / ml fibroblast growth factor-2 and 1-20 ng / ml epidermal growth factor (EGF) were added; It may also be a medium in which 10 wt% fetal calf serum, 0.1 mM 2-mercaptoethanol, 1 wt% MEM non-essential amino acids, 100 U / ml penicillin and 100 μg / ml streptomycin are added. More preferably the proliferation medium is (i) 4 ng / ml fibroblast growth factor-2 or (ii) 10 ng / ml epidermal growth factor or (iii) 4 ng / ml fibroblast growth factor in the basal medium. -2 and 10 ng / ml epidermal growth factor (EGF) are added; Medium with 10 wt% fetal calf serum, 0.1 mM 2-mercaptoethanol, 1 wt% MEM non-essential amino acid, 100 U / ml penicillin and 100 μg / ml streptomycin.

According to another aspect of the invention, the step of propagating amniotic fluid-derived mesenchymal stem cells according to the method; And (b) encapsulation of the amniotic fluid-derived mesenchymal stem cells obtained from step (a) in a fibrin hydrogel comprising transforming growth factor-β3 (TGF-β3). There is provided a method for producing a transplant structure for cartilage generation in vivo, comprising the step of forming a transplant structure).

According to the present invention, when culturing amniotic fluid-derived mesenchymal stem cells in the presence of fibroblast growth factor-2 (FGF-2) and / or epidermal growth factor (EGF), It has been found that amniotic-derived mesenchymal stem cells can be proliferated stably for long periods of time without chromosomal abnormalities. In addition, it has been confirmed that the amniotic fluid-derived mesenchymal stem cells thus proliferated can be effectively differentiated into chondrocytes in vivo. It can be usefully used to prepare implantable constructs.

FIG. 1 shows the results of evaluating time spent in each passage while subcultured hAF-MSCs in the presence of FGF2, EGF, or FGF2 + EGF. In FIG. 1, growth-A indicates passage in the presence of FGF2, growth-B in the presence of FGF2 + EGF, growth-C in the presence of EGF, and growth-D indicates passage in the conventional culture method.
Figure 2 shows the results of analyzing the cell number and the chromosome in the state incubated the same amount of hAF-MSCs in the sixth passage for 24 hours under each condition.
3 shows the results of FACS analysis of MSC characteristics at the sixth passage. CD166, CD29, and CD44 were measured as mesenchymal positive markers and CD45 and CD34 as negative markers.
4 shows cartilage differentiation of AF-MSCs, AD-MSCs, and BM-MSCs cultured in vitro as measured by RT-PCR and real time--QPCR. A: RT-PCR analysis of COLII, SOX 9, Aggrecan, COMP, and COL I gene expression. Total RNA was obtained from MSCs cultured for 21 days in fibrin hydrogels with or without TGF-β3 (100 ng / ml). B: Real-time--qPCR analysis of cartilage gene expression levels. a: COL II, b: SOX 9, c: aggrecan, d: COMP, and e: COL I gene expression. Data represent mean ± SD (n = 3). *, p <0.05.
5 is a result of measuring the total collagen content in the cartilage (neo-cartilage) formed by the differentiation of hMSCs mixed with fibrin hydrogel loaded with TGF-β3. Three weeks of in vitro culture were analyzed by Masson's trichrome (A), GAG / DNA content (B), and western blotting (C). A: a, c, & e: fibrin hydrogel with TGF-β3, b, d, & f: fibrin hydrogel without TGF-β3. a & b: AF-MSCs, c & d: AD-MSCs, e & f: BM-MSCs.
Figure 6 shows the results of histological analysis of the cartilage (neocartilage) formed by hMSCs after culturing hMSCs mixed with fibrin hydrogel loaded with TGF-β3 for 3 weeks. Cultures incubated in vitro for 3 weeks were replaced with H & E (af), alcian Analysis was by blue (gl) and safranin-O (mr) staining. (a, c, e, g, i, k, m, o, & q) hMSCs alone; (b, d, f, h, j, l, n, p & r) hMSCs + TGF-β3. (a, b, g, h, m, & n) AF-MSCs; (c, d, I, j, o, & p) AD-MSCs; (e, f, k, l, q, & r) BM-MSCs. Bars represent 100 mm.
Figure 7 shows the immunohistochemical analysis of the cartilage (neocartilage) formed by hMSCs mixed in the presence or absence of TGF-β3 in the fibrin hydrogel. Cultures cultured in vitro for 3 weeks were analyzed by DAB staining. (a, b, e, f, i, & j) COL I; (c, d, g, h, k, & l) COL II. (a, c, e, g, i, & k) hMSCs alone; (b, d, f, h, j, & l) hMSCs + TGF-β3. (ad) AF-MSCs, (eh) AD-MSCs, & (il) BM-MSCs. Bars represent 100 mm.
8 shows the results of analysis of cartilage differentiation of AF-MSC, AD-MSC, and BM-MSC cultured in vitro using RT-PCR and real-time-QPCR. A: RT-PCR analysis of COL II, SOX 9, Aggrecan, COMP, and COL I gene expression. Total RNA was obtained from MSCs grown for 21 days on fibrin hydrogels with or without TGF-β3 (100 ng / ml). GAPDH was used as internal control. B: Real-time-qPCR analysis of cartilage expression levels. a: COL II, b: SOX 9, c: aggrecan, d: COMP, & e: COL I gene expression. Data represent mean ± SD (n = 3). *, p <0.05.

The present invention relates to (i) fibroblast growth factor-2 (FGF-2) or (ii) epidermal growth factor (EGF) or (iii) fibroblast growth factor-2 in basal medium. Provided is a method of proliferating amniotic fluid-derived mesenchymal stem cells, comprising culturing amniotic fluid-derived mesenchymal stem cells in a growth medium comprising (FGF-2) and epidermal growth factor (EGF).

The amniotic fluid-derived mesenchymal stem cells are known methods, for example, separation methods established by De Coppi et al. (De Coppi, P., Bartsch, G., Jr., Siddiqui, MM, Xu, T., Santos , CC and Perin, L., et al., Isolation of amniotic stem cell lines with potential for therapy, Nat Biotechnol 25 (1), 2007, 100-106). Amniotic-derived mesenchymal stem cells can be obtained from amniotic fluids derived from humans in vitro, and amniocentesis, which is typically performed on prenatal pregnant women, for early diagnosis of hereditary genetic diseases. It can be separated from the amniotic fluid separated in vitro through the known method.

As a basal medium of the growth medium of the present invention, a known medium commonly used for cell culture may be used. For example, a commercially available DMEM / F12 medium (Gibco BRL), α-MEM medium ( Gibco, Invitrogen) and the like can be used, but is not limited thereto. In one embodiment, DMEM / F12 medium (Gibco BRL) can be used as the basal medium.

The growth medium used in the growth method according to the present invention comprises fibroblast growth factor-2 (FGF-2) and / or epidermal growth factor (EGF) in the basic medium, more preferably fiber Blast growth factor-2 (FGF-2).

In the growth method according to the present invention, the growth medium is added to (i) fibroblast growth factor-2 or (ii) epidermal growth factor or (iii) fibroblast growth factor-2 and epidermal growth factor to the basal medium; It may also be a medium to which fibroblast growth factor-2, fetal bovine serum, 2-mercaptoethanol, MEM non-essential amino acids, and antibiotics are added.

In one embodiment, the proliferation medium is (i) 1-10 ng / ml fibroblast growth factor-2 or (ii) 1-20 ng / ml epidermal growth factor or (iii) 1-10 ng in basal medium. / ml fibroblast growth factor-2 and 1-20 ng / ml epidermal growth factor (EGF) were added; It may also be a medium in which 10 wt% fetal calf serum, 0.1 mM 2-mercaptoethanol, 1 wt% MEM non-essential amino acids, 100 U / ml penicillin and 100 μg / ml streptomycin are added. More preferably the proliferation medium is (i) 4 ng / ml fibroblast growth factor-2 or (ii) 10 ng / ml epidermal growth factor or (iii) 4 ng / ml fibroblast growth factor in the basal medium. -2 and 10 ng / ml epidermal growth factor (EGF) are added; Medium with 10 wt% fetal calf serum, 0.1 mM 2-mercaptoethanol, 1 wt% MEM non-essential amino acid, 100 U / ml penicillin and 100 μg / ml streptomycin.

The present invention also comprises the steps of propagating amniotic fluid-derived mesenchymal stem cells according to the above method; And (b) encapsulation of the amniotic fluid-derived mesenchymal stem cells obtained from step (a) in a fibrin hydrogel comprising transforming growth factor-β3 (TGF-β3). Or embedding) to provide a method for producing a transplant structure for cartilage generation in vivo, comprising the step of forming a transplant structure.

Forming the implantable construct (ie, step (b)) is the prior art of the inventors (e.g. Park, JS, Yang, HN, Woo, DG, Jeon, SY and Park, KH, Chondrogenesis of human mesenchymal stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models, Biomaterials 32 (6), 2011, 1495-4507). For example, the amniotic fluid-derived mesenchymal stem cells obtained by performing the proliferation step as described above are suspended in fibrinogen solution and then homogenized with aprotinin, thrombin, fibrin-stabilizing factor XIII, and 50 mM CaCl _2. The resulting mixture can then be dropped to form a gel, thereby forming a fibrin hydrogel. As is clear from the following test examples, when transplanting the transplant construct obtained as described above in vivo, amniotic fluid-derived mesenchymal stem cells encapsulated in fibrin hydrogel in vivo differentiate into cartilage. Therefore, the implant for construct according to the present invention can be usefully used as a cell therapy for in vivo cartilage production.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Amniotic-derived Intermediate lobe  Stem Cell Proliferation and Evaluation

(1) human amniotic fluid-derived Intermediate lobe  Stem Cells( hAF - MSCs )

Human amniotic fluid-derived mesenchymal stem cells (hAF-MSCs) are described by De Coppi et al. (De Coppi, P., Bartsch, G., Jr., Siddiqui, MM, Xu, T., Santos, CC and Perin, L). , et al., Isolation of amniotic stem cell lines with potential for therapy, Nat Biotechnol 25 (1), 2007, 100-106). The amniotic fluid was used by a pregnant woman who had undergone amniotic fluid testing for chromosome test, and received extra cells remaining after the test from the genetic laboratory of Gangnam Tea Hospital.

(2) human amniotic fluid-derived Intermediate lobe  Stem Cells( hAF - MSCs Proliferation of

HAF-MSCs harvested by trypsinization (10% FBS, 2-mercaptoethanol (Gibco BRL), 1% MEM non-essential amino acid solution (Gibco BRL), 100 U / ml penicillin (Gibco BRL), In DMEM / F12 medium (Gibco BRL) containing 100 μg / ml streptomycin (Gibco BRL), and 4 ng / ml FGF2 (Invitrogen), were grown under conditions of 5% CO ₂ at 37 ° C. hAF-MSCs were 1 The medium was changed once every 3 or 4 days with dilution of 4 to 1: 6, and every time the cells were filled to about 70% to 90% of the culture vessel surface (ie about 70% to 90% confluence), Subculture was performed (hereinafter, referred to as 'proliferation-A').

In addition, a medium further containing 10 ng / ml of EGF in the growth medium (hereinafter referred to as 'proliferation-B'); Alternatively, hAF-MSCs were grown in the same manner as above in a medium containing 10 ng / ml EGF instead of FGF2 (hereinafter, referred to as 'proliferation-C').

In addition, for evaluation of proliferation rate, De Coppi et al. (De Coppi, P., Bartsch, G., Jr., Siddiqui, MM, Xu, T., Santos, CC and Perin, L., et al., Isolation of amniotic stem cell lines with potential for therapy, medium according to Nat Biotechnol 25 (1), 2007, 100-106, i.e. 15% ES-FBS, 1% glutamine, 18% Chang B (Irvine Scientific), 2% Chang Amniotic-derived mesenchymal stem cells were cultured using C (Irvine Scientific) and α-MEM medium (Gibco, Invitrogen) added with 1% penicillin / streptomycin (hereinafter referred to as 'proliferation-D'). ).

(3) human amniotic fluid-derived Intermediate lobe  Stem Cells( hAF - MSCs Proliferation evaluation of

In (2), while continuing passage in each condition (that is, growth-A to growth-D), the results of evaluating the time required for each passage are as shown in FIG. 1. Furthermore, in (2), hAF-MSCs exhibited a confluency of 70-90% while subcultured under the conditions of proliferation-A, proliferation-B, and proliferation-D under conditions 6-24. As a result of measuring the time taken for passage and the number of passages continued to grow, the results are as shown in FIG. 1, and the results of observing the number of proliferation when culturing for 24 hours in 6 passages and analyzing the chromosomes are shown in FIG. 2.

From the results of FIG. 1, it can be seen that when the hAF-MSCs were cultured in the presence of FGF2 and / or EGF according to the present invention, passage was further progressed and the time to passage was shortened. In particular, it can be seen that proliferation-A [FGF alone, purple] and proliferation-B [FGF + EGF, green] are more effective. In addition, it can be seen from the results of FIG. 2 that when the hAF-MSCs are propagated in the presence of FGF2 or FGF2 and EGF according to the present invention, the growth of cells is fast, the shape is excellent, and the chromosome state is stable.

Example  2. Proliferated Human Amniotic Fluid-Derived Intermediate lobe  Stem Cells( hAF - MSCs ) And Eruption  evaluation

Characterization and differentiation of human amniotic fluid-derived mesenchymal stem cells (hAF-MSCs) propagated according to Example 1 (proliferated under the proliferation-A condition) are as follows: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) ) And human adipose derived mesenchymal stem cells (hAD-MSCs).

(1) Materials and test methods

<1> cell harvest and culture

Human bone marrow mesenchymal stem cells (hBM-MSCs) were purchased from Cambrex (Cambrex, East Rutherford, NJ, USA), 10% fetal calf serum (FBS), 100 U / ml penicillin G, 100 mg / ml streptomycin , And cultured in α-MEM medium (Gibco BRL, Grand Island, NY, USA) to which 0.25 mg / ml amphotericin B was added (lot no .: 7F3674, 7F3675, and 7F3677). During incubation, non-adherent cells were removed by changing medium and the medium was changed every 3 days. After one week of primary culture, cells reached 80% confluence and cells were detached and passaged using 0.25% trypsin / EDTA (Gibco BRL) solution. Human adipose derived mesenchymal stem cells (hAD-MSCs) were provided by Asan Hospital.

<2> Flow cell  Cell characterization by analysis

Cells were harvested by trypsinization, washed and centrifuged at 300 g for 5 minutes. The cells were counted and 5 × 10 ⁵ cells were placed in a fluorescence-activated cell sorting (FACS) polypropylene tube. Flow cytometry was performed using the following monoclonal antibodies: anti-human CD29, CD34, CD44, CD45, and CD166 (Santa Cruz Biotechnology, Santa Cruz, CA). Cells were resuspended in phosphate-buffered saline (PBS) at a concentration of 1 × 10 ⁶ cells / 200 μl and incubated with 20 μl of antibody at 4 ° C. for at least 1 hour. Antibodies were conjugated to fluorescent isothiocytate (FITC) and specific for human markers associated with the mesenchymal lineage. At the point where the use of the secondary antibody was needed, the cells were incubated in the dark for 30 minutes at 4 ° C. in the secondary antibody to allow binding to the primary antibody. Flow cytometry was performed using Cell Quest software and FACSCalibur instrument (BD Biosciences). Data was analyzed using Cell Quest software (BD Biosciences). The results are expressed as mean ± standard deviation. Differences were considered statistically significant at P <0.05.

<3> Preparation of Fibrin Gel Structure Containing Growth Factor

For preparation of fibrinogen gels containing control gels or TGF-β3 (final concentration 100 ng / ml) (n = 5), hMSCs were collected by centrifugation, followed by fibrinogen solution (18 mg / ml). ml (10 wt.%), Mokam Research Institute, Suwon, South Korea). Each 1 × 10 ⁶ cells / ml suspension was diluted with aprotinin (Mount Cancer Research Institute), 60 U / ml thrombin (1000 U / mg protein: Sigma, St. Louis, MO, USA), fibrin-stabilizing factor XIII, and 50 Homogenized with mM CaCl ₂ . 250 μl of fibrin mixture was then dropped into a 5 ml polypropylene round bottom tube to form a gel. Each gel was transferred to a new 5 ml polypropylene round bottom tube and incubated in α-MEM containing 1% antibiotic (100 mg / ml streptomycin, 100 IU / ml penicillin). Controls were prepared using the same method.

<4> total collagen and glycosaminoglycans ( GAG ) analysis

DNA and GAG analyzes were performed three times for each test group and control group at each time point. Cell counts were determined using PicoGreen assay (Molecular Probes, Eugene, OR) by measuring the double-stranded DNA content according to the manufacturer's instructions. The fluorescence of the negative, cell-free control group was subtracted from the fluorescence value of the test group. PicoGreen assay was also used to determine total DNA content. Similarly, GAG content was measured using a dimethylmethylene blue dye (DMMB) assay. That is, 50 ml of the sample digested with papain was incubated with 2 ml of DMMB dye, and the absorbance at 520 nm was measured with a spectrophotometer. Shark chondroitin sulfate C (Sigma) was used as a standard. Total GAGs were normalized to total DNA.

Samples obtained from nude mice were digested with papain digestion solution (125 μg / mL papain, 5 mM L-cysteine, 100 mM Na ₂ HPO ₄ , 5 mM EDTA, pH 6.8) for 16 hours at 60 ° C., followed by Total collagen was measured. Sirius red was dissolved in a saturated solution of picric acid (1.3%, Sigma) at a concentration of 1 mg / mL to prepare a dye solution (pH 3.5). The digested samples were dried at 37 ° C. for 24 h in 96-well plates and reacted with the dye solution on a stirrer for 1 h. The sample was washed 5 times with 0.01 N HCl and resuspended in 0.1 N NaOH. Absorbance was measured at 550 nm using an ELISA Reader (BIO-TEK Instruments Inc., Winooski, VT). Finally, the total collagen amount of each sample was calculated by comparison with the standard curve of 0-10 μg / ml bovine collagen (Sigma).

<5> RNA  Extraction and Reverse transcription - PCR  ( RT - PCR )

RNA extraction was performed using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA pellets were dissolved in 20 μl of RNase-free and DN-free purified water and RNA yield was determined by measuring optical density at 260 nm. Total RNA was measured using a NanoDrop spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE). Total RNA (0.5 μg) was reverse transcribed with 20 μl of reaction volume using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase and random hexamers according to the manufacturer's (Invitrogen) instructions. Each RT-PCR reaction used 1 μl of cDNA. PCR reactions were performed using 2X SeeAmp ™ ACP ™ Master Mix II (Seegene, Seoul, Korea) under the following conditions: 1 cycle for 10 minutes at 94 ° C, then 30 seconds at 94 ° C, 45 seconds at 58 ° C, 72 35 cycles of 45 seconds at &lt; RTI ID = 0.0 &gt; The PCR product was electrophoresed on a 1.5% agarose gel and then visualized by ethidium bromide staining. The expression of the following genes was measured by real time QPCR with ExiCycler (Bioneer, Daejeon, Korea, http://www.bioneer.com): collagen form II (COL II), SOX9, aggrecan and cartilage oligomeric protein , COMP). Each RT reaction (1 μl) was followed by 2.0 mM MgCl ₂ , 0.5 μM of each primer, and 2 × SYBR? Amplified in 20 μl PCR assay volume containing Green PCR Master Mix. Samples in ExiCycler were subjected to 45 cycles of initial denaturation at 94 ° C. for 10 minutes, then 10 seconds at 94 ° C., 30 seconds at 58 ° C. and 30 seconds at 72 ° C. Relative amounts of specific genes were determined via the 2-ΔΔCT method according to the manufacturer's recommendations. To confirm the amplification of specific transcripts, melting curve profiles were created at the end of each PCR by cooling the sample to 40 ° C. and then slowly heating to 95 ° C. while continuously monitoring the fluorescence. Primers used in this study are shown in Table 1 below.

PCR primers and product size gene SEQ ID NO: order Size (bp) SOX-9 One sense 5'-TTCATGAACTTGACCGACGA-3 ' 290 2 Antisense 5'-CACACCATGTTCGCGTTCAT-3 ' Col ii 3 sense 5'-GCACCCATGGACATTGGAGGG-3 ' 366 4 Antisense 5'-GACACGGAGTAGCACCATCG-3 ' COMP 5 sense 5'-CAGGACGACTTTGATGCAGA-3 ' 360 6 Antisense 5'-AAGCTGGAGCTGTCCTGGTA-3 ' Aggrecan 7 sense 5'-CCTTGGAGGTCGTGGTGAAAGG-3 ' 280 8 Antisense 5'-AGGTGAACTTCTCTGGCGACGT-3 ' Col i 9 sense 5'-AGAACATCACCTACCACTGC-3 ' 307 10 Antisense 5'-ATGTCCAAAGGTGCAATATC-3 ' GAPDH 11 sense 5'-TCACAATCTTCCAGGAGCGA-3 ' 366 12 Antisense 5'-CACAATGCCGAAGTGGTCGT-3 '

<6> nude mouse transplant

Animal testing was approved by the Animal Care Committee of CHA University. BALB / c female mice (6 weeks old) were divided into three groups. Group I (n = 16) was injected subcutaneously in the back of each mouse with a fibrin hydrogel with or without TGF-β3 mixed with AF-MSCs. Group II (n = 16) was injected subcutaneously in the back of each mouse with a fibrin hydrogel with or without TGF-β3 mixed with AD-MSCs. Group III (n = 16) was injected subcutaneously in the back of each mouse by mixing fibrin hydrogels with or without TGF-β3 with BM-MSCs. One to four weeks after treatment, mice were lethal by injection of an anesthetic in excess (n = 4 at each time point), and the skin at the site of injection (2 × 2 cm ² ) was carefully cut and used for subsequent biological tests. Photographs of skin flaps were taken to record the appearance of tissue around the treated area.

<7> Western Blot  analysis

Cells were lysed in RIPA buffer (Pierce, Rockford, IL) to which a complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) was added. About 30-50 mg of protein was then loaded onto a 10-15% SDS polyacrylamide gel (SDS-PAGE) and transferred to Immobilon-P (Millipore Corp., Bedford, Mass.). The membrane was blocked in 5% skim milk in Tris-buffered saline (TBS) -Tween 20 (0.02%) and incubated with the following primary antibody: anti-aggrecan (Abcam, Cambridge, UK), Anti-type 2 collagen, anti-SOX-9 (Chemicon, Temecula, Calif.) And anti-β-actin (Sigma). Finally, the blots were developed by chemiluminescence using Amersham ECL reagent (GE Healthcare, Little Chalfont, UK).

<8> Immunohistochemistry

The injection site was completely cut out and then processed for histological analysis. In other words, samples taken at each time point were collected from O.C.T. It was embedded in a compound (TISSUE-TEKs 4583, Sakura Finetek USA, Inc.) and frozen. The fibrin construct seeded with cells was then soaked in 4% paraformaldehyde solution inoculated for 30 minutes to fix the construct to the slide. Each sample was cut thin at −20 ° C. to 10 mm thickness and stained with hematoxylin and eosin (H & E). Hydrated cryosections were stained with safranin-O and alcian blue to perform histological analysis. Masson's trichrome staining was used for the detection of collagen fibers. In this method, the collagen fibers are dyed blue, the nuclei are dark reddish brown, and the cytoplasm is reddish pink.

Immunohistologic analysis was performed using the following primary antibodies: anti-SOX9 (Chemicon, Temecula, CA. Dilution factor 1: 500), anti-type 1 collagen and anti-type 2 collagen (Chemicon, Temecula, CA) Dilution factor 1: 500). All incubations were performed in a humid chamber. In vitro In vitro ): formalin-fixed paraffin embedded sections were deparaffinized and rehydrated, followed by 15 minutes in 3% H ₂ O ₂ (Fisher, Ottawa, ON, Canada). Antigen retrieval was performed by incubation followed by incubation with acidic citric acid buffer as described above. The pieces were incubated for 30 minutes in anti-type 1 collagen or anti-type 2 collagen, followed by 45 minutes in 5% normal goat serum (Invitrogen, Carlsbad, Calif.). The slides were washed, incubated with biotinylated secondary antibody (1: 500; Invitrogen) for 30 minutes and then incubated with AEC chromogen (Invitrogen) for 15 minutes. The pieces were then counterstained with Mayer's hematoxylin (Sigma-Aldrich). In vitro vivo ) Samples were embedded in OCT compounds (TISSUE-TEKs 4583, Sakura Finetek USA, Inc), frozen and fragmented. The pieces were washed with PBS and incubated with the primary antibody described above. FITC or RITC-conjugated secondary antibody (Molecular Probes, USA) was added followed by three washes with PBS and 5 with 4 ', 6-diimidino-2-phenylindole (DAPI, Molecular Probes) stain. Incubate for minutes. Finally, the pieces were analyzed using confocal laser scanning microscope.

<9> Statistical Analysis

Statistical analysis was performed using the students t- test. A value of p <0.05 was considered statistically significant.

(2) results

<1> different kinds hMSCs of Immunophenotypic ( Immunophenotypic Characteristics

To confirm that the stem cell phenotype is maintained in subculture, the morphology of the mesenchymal stem cells was evaluated by flow cytometry. Mesenchymal properties were assessed using CD166, CD29 and CD44 as positive MSC markers and CD45 and CD34 as negative MSC markers. MSC markers CD166, CD29 and CD44 were observed in at least 90% of cells, whereas MSC negative markers CD45 and CD34 were observed in less than 10% of cells (FIG. 3).

<2> In vitro  Cultured hMSCs of RT - PCR  analysis

Cartilage differentiation of chondrogenic differentiation of hMSCs was analyzed by RT-PCR based on the expression of cartilage differentiation marker mRNA including COL II, aggrecan, COMP, and SOX9 (FIG. 4A). BM-MSCs, AD-MSCs, and AF-MSCs showed different expression patterns for cartilage differentiation markers. As a result of the PCR analysis, COL II, SOX9, aggrecan, and COMP were expressed higher in BM-MSCs than in AD-MSCs and AF-MSCs. However, several cartilage-specific genes such as aggrecan and COMP were expressed at similar levels in all three hMSCs. In contrast, the bone differentiation marker COL I was expressed at low levels in all hMSCs stimulated by TGF-β3.

When specific mRNAs involved in cartilage differentiation were quantified by real time-qPCR, COL II, aggrecan, SOX9, and COMP were produced by all hMSCs, irrespective of whether they were stimulated by TGF-β3. lost. In addition, the bone differentiation marker COL I was not expressed (FIG. 4B). In the control group (TGF-β3 untreated), all of the stem cells in the untreated fibrin hydrogel showed little chondrocyte-related markers, whereas all hMSCs in the fibrin hydrogel containing TGF-β3 were chondrocyte-associated. Increased expression of the marker was shown. The expression levels of COL II and SOX9 were 20-30 fold higher than the control. The expression levels of aggrecan and COMP genes were increased by 4 times in AF-MSCs, 8 times in AS-MSCs, and 8 times in BM-MSCs when treated with TGF-β3. However, bone differentiation marker COL I was not expressed at all in three different hMSCs stimulated with TGF-β3.

<3> In vitro  Collagen, GAG , And measurement of cartilage specific proteins

Total collagen production of hMSCs encapsulated in hydrogel constructs, with or without TGF-β3, was measured by Masson's Trichrome staining (FIG. 5A). BM-MSCs, AD-MSCs, and AF-MSCs encapsulated in fibrin hydrogel constructs containing TGF-β3 are BM-MSCs,, AD-MSCs, and AF- encapsulated in hydrogels containing no TGF-β3. More collagen was produced compared to MSCs.

In the presence or absence of TGF-β3, GAG production by hMSCs encapsulated in hydrogel constructs was measured as a function of DNA content (FIG. 5B). Encapsulated hMSCs showed significantly increased GAG production in response to the addition of TGF-β3. In addition, GAG / DNA levels in BM-MSCs, AD-MSCs, and AF-MSCs treated with TGF-β3 were higher than M-MSCs, AD-MSCs, and AF-MSCs not treated with TGF-β3. 6.4, 5.8, and 5.2 times higher, respectively. However, there was no significant difference in the GAG content of BM-MSCs, AD-MSCs, and AF-MSCs treated with TGF-β3.

Western blot analysis was used to determine whether TGF-β3 stimulates cartilage specific protein expression in hMSCs encapsulated in fibrin hydrogel constructs. Cartilage specific proteins were secreted by hMSCs encapsulated in fibrin hydrogel constructs (FIG. 5C), which meant that encapsulated hMSCs differentiated into chondrocytes. Cartilage specific proteins, such as aggrecan, COL II, and SOX9, were found only in hMSCs stimulated by the addition of TGF-β3.

<4> hMSCs Histological examination

To confirm that encapsulated hMSCs are present in the fibrin hydrogel, histological methods were used to assess stem cell morphology, distribution of encapsulated cells, and differentiation of encapsulated hMSCs into chondrocytes (FIG. 6). Cells in fibrin hydrogels without TGF-β3 were mostly undifferentiated hMSCs, although small amounts of differentiated cells were detected. HMSCs in fibrin hydrogels containing TGF-β3 showed predominantly differentiated cell morphology (Figure 6. b, d, & f).

In the production of cartilage in natural tissues, lacunae formation, a marker of the form of cartilage tissue, is found. To confirm that hMSCs differentiate into chondrocyte form by the addition of TGF-β3, samples were evaluated using safranin-O and alcian blue staining (FIG. 6. gr). HMSCs cultured in vitro in the presence of TGF-β3 differentiated into chondrocytes, and these chondrocytes clearly and deeply showed blue and orange staining indicating polysaccharides and proteoglycans, respectively, and these properties were evident in all hMSCs evaluated (FIG. 6 h, j, l, n, p, & r).

<5> COL  I and II Immunohistochemical analysis

In hMSCs encapsulated in fibrin hydrogels with or without TGF-β3, cartilage differentiation of hMSCs was confirmed immunohistochemically based on the detection of specific proteins such as COL II. Immunostaining with COL II antibodies was used to detect COL II secreted from encapsulated hMSCs (FIG. 7. c, d, g, h, k & l). As shown in Figures d, h, & l, hMSCs treated with TGF-β3 were strongly stained for COL II. COL I, a bone differentiation marker, was hardly detected in hMSCs in fibrin hydrogels with TGF-β3 (Fig. 7. b, f, & j). In addition, all hMSCs cultured in the absence of TGF-β3 did not stain well for COL II, but some COL I staining was observed (Fig. 7. c, g & k).

<6> implanted hMSCs Nude mouse model for cartilage differentiation

To confirm in vitro cartilage differentiation of different types of hMSCs, chondrocyte-associated specific genes were detected by RT-PCR and real time--qPCR (FIGS. 8A & B). As shown in FIGS. 8A and 8B, in all hMSCs in fibrin hydrogels containing TGF-β3, high expression of specific genes of COL II, SOX9, aggrecan, and COMP compared to the control group (without TGF-β3). It became. AF-MSCs showed 40-fold gene expression compared to control (AF-MSCs: 4 ± 0.6 / control: 0.1 ± 0.02). AS-MSCs showed higher aggrecans expression compared to the control (AS-MSCs: 3.7 ± 0.4 / control: 0.2 ± 0.02). High aggrecan expression was observed in BM-MSCs (5.4 ± 0.7) (FIG. 8. Bc), but no difference was found in the control (control: 1.8 ± 0.05). In addition, in the detection of COMP genes, all hMSCs showed more than 50-fold compared to the control (Fig. 7. B d). COL II and SOX9 gene expression from hMSCs treated with TGF-β3 showed similar results (FIG. 8. B a & b). However, COL I, a bone differentiation marker liberated from hMSCs in fibrin hydrogels containing TGF-β3, was hardly detected.

(3) Discussion and conclusion

In this study, hMSCs with different origins were measured using positive and negative markers and the like to evaluate their usefulness as a cell stock for clinical application. For phenotypic determination of hMSCs, proliferated cells were evaluated by flow cytometry. More than 90% of BM-MSC, AD-MSC, and AF-MSC cells were positive for the mesenchymal markers CD166, CD29, and CD44, while less than 10% were positive for the non-mesenchymal markers CD45 and CD34 ( 3). These results indicate that the cells retained the mesenchymal phenotype even after passage.

In addition, cartilage differentiation capacity was tested in vitro and in vivo for hMSCs of different origin encapsulated in a hydrogel containing TGF-β3. TGF-β3, as a cartilage differentiation stimulant, was induced in injectable hydrogels to induce proliferation and differentiation of hMSCs. By co-encapsulation of TGF-β3 and hMSCs, the differentiation capacity of BM-MSC, AD-MSC, and AF-MSC was markedly increased compared to the control (TGF-β3 not present) (Fig. 4. A & B), There was a slight difference between the stem cells. SOX9 and COMP genes were highly expressed in AF-MSCs, but COL II and aggrecan were not highly expressed. COL I, a bone differentiation marker, was not significantly expressed in the hMSCs. These results indicate that the cartilage differentiation capacity between hMSCs with different origins is different.

When comparing gene expression between BM-MSCs, AD-MSCs, and AF-MSCs cultured with or without TGF-β3, the typical cartilage-specific marker proteins COL II, aggrecan, SOX9, and COMP are TGF- In fibrin hydrogels containing β3, they were highly expressed by cells. In addition, RT-PCR and Western blotting analysis showed that BM-MSCs and AD-MSCs express higher levels of markers as compared to AF-MSCs. These results suggest that expression of cartilage-specific proteins is stimulated by TGF-β3 and that fibrin hydrogels containing TGF-β3 are essential for stimulating the expression of certain cartilage differentiation markers after hMSCs transplantation. In addition, the cartilage differentiation capacity of AF-MSCs was lower than that of BM-MSCs and AD-MSCs.

To assess the ability of transplanted hMSCs to differentiate into cartilage-like cells in vivo, RT-PCR analysis was performed on cartilage-specific differentiation markers COL II, aggrecan, SOX9 and COMP in transplanted hMSCs. As shown in FIGS. 7A and 7, these genes were expressed higher in hMSCs encapsulated in fibrin hydrogels containing TGF-β3 as compared to hMSCs in fibrin hydrogels without TGF-β3.

Histological evaluation of cartilage differentiation showed that the amount of damaged tissue regeneration from transplanted hMSCs differentiated into cartilage-like cells increased when mixed with TGF-β3 in fibrin hydrogels. Typical morphological changes from fibroblast-like cells to cells forming lacunae have been shown. At the same time accumulation of proteoglycans and polysaccharides was detected in samples from hydrogels containing TGF-β3. HMSCs encapsulated in a hydrogel containing no TGF-β3 produced only a few extracellular matrices. These results indicate that the hMSCs studied are suitable as a source of cells for cell therapy.

In conclusion, hMSCs mixed with TGF-β3, encapsulated in fibrin hydrogel, produced differentiated chondrocytes. Evaluation of hMSCs derived from different origins, using RT-PCR, real-time-QPCR, histological analysis and immunohistochemical analysis, showed that when they were cultured in vitro and implanted in vivo in the presence of TGF-β3, It has been shown to express high levels of specific genes and proteins. These results indicate that all of the hMSCs evaluated in this study can be used for stem cell therapy because they have the ability to differentiate into specific cell types in the presence of TGF-β3.

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Claims (5)

(I) fibroblast growth factor-2 (FGF-2) or (ii) epidermal growth factor (EGF) or (iii) fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) in basal medium. A method of propagating amniotic fluid-derived mesenchymal stem cells, comprising culturing the amniotic fluid-derived mesenchymal stem cells in a growth medium comprising. The method of claim 1, wherein the growth medium is added to (i) fibroblast growth factor-2 or (ii) epidermal growth factor or (iii) fibroblast growth factor-2 and epidermal growth factor to the basal medium; In addition, fibroblast growth factor-2, fetal bovine serum, 2-mercaptoethanol, MEM non-essential amino acids, and the method of proliferation of amniotic fluid-derived mesenchymal stem cells, characterized in that the medium is added. The growth medium according to claim 2, wherein the growth medium is (i) 1-10 ng / ml fibroblast growth factor-2 or (ii) 1-20 ng / ml epidermal growth factor or (iii) 1-10 ng / ml fibroblast growth factor-2 and 1-20 ng / ml epidermal growth factor (EGF) are added; Amniotic fluid, characterized in that 10% by weight fetal calf serum, 0.1 mM 2-mercaptoethanol, 1% by weight MEM non-essential amino acids, 100 U / ml penicillin and 100 μg / ml streptomycin added Method of proliferation of derived mesenchymal stem cells. 4. The growth medium according to claim 3, wherein the proliferation medium is (i) 4 ng / ml fibroblast growth factor-2 or (ii) 10 ng / ml epidermal growth factor or (iii) 4 ng / ml fibroblasts in the basal medium. Growth factor-2 and 10 ng / ml epidermal growth factor (EGF) are added; Amniotic fluid-derived medium characterized by addition of 10% fetal calf serum, 0.1 mM 2-mercaptoethanol, 1% by weight MEM non-essential amino acids, 100 U / ml penicillin and 100 μg / ml streptomycin Mesenchymal stem cell proliferation method. (a) propagating amniotic fluid-derived mesenchymal stem cells according to any one of claims 1 to 4; And (b) encapsulating the amniotic fluid-derived mesenchymal stem cells obtained from step (a) in a fibrin hydrogel comprising transforming growth factor-β3 (TGF-β3) to form a construct for transplantation. Method for producing a transplant construct for in vivo cartilage production.

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