KR100956371B1 - In vitro method of culturing adipose-derived stromal cells for cardiomyogenic differentiation - Google Patents

Abstract

The present invention relates to an in vitro culture method for differentiating adipose derived stromal cells into cardiomyocytes. More specifically, the present invention provides a method for culturing fat-derived stromal cells comprising adding TGF-β1 to adipocyte-derived stromal cells in vitro and differentiating them into cardiomyocytes and myocardium derived from fat-derived stromal cells differentiated by the method. It is about a cell population.

The culture method and the myocardial cell population according to the present invention increase the practicality of ADSCs as a transplant cell therapy that can be used for the treatment of myocardial infarction, and are useful for the production of such therapeutic agents. Pre-differentiation of ADSCs into cardiomyocytes using this method prior to transplantation is useful for improving the effect of treating myocardial infarction.

Adipose-derived stromal cells, Adipose-derived stem cells, Cardiomyocyte differentiation, TGF

Description

In vitro culture of adipose derived stromal cells for differentiation into cardiomyocytes {IN VITRO METHOD OF CULTURING ADIPOSE-DERIVED STROMAL CELLS FOR CARDIOMYOGENIC DIFFERENTIATION}

The present invention relates to an in vitro culture method for differentiating adipose derived stromal cells into cardiomyocytes. More specifically, the present invention provides a method for culturing fat-derived stromal cells comprising adding TGF to adipocyte-derived stromal cells in vitro and differentiating them into cardiomyocytes and a population of cardiomyocytes derived from adipose-derived stromal cells differentiated by the method. It is about.

Stem cell transplantation has been extensively studied as a treatment for regenerating myocardial tissue damaged by myocardial infarction. Embryonic stem cells, bone marrow-derived mesenchymal stem cells (BMMSCs), umbilical cord blood cells, and adipose-derived stromal cells (ADSCs) are also called cardiomyocytes. Differentiable (Cho, SW, et al., 2006, Biochem. Biophys . Res . Commun . 340 : 573-582; Li, TS, et al., 2005, Circulation 111 : 2438-2445 .; Makino, S., et al., 1999, 103 : 697-705 .; Orlic, D., et al., 2001, Ann . NY . Acad. Sci . 938 : 221-229 .; Yamada, Y., et al., 2007, Stem Cells 25 : 1326-1333). Clinical studies of self-derived BMMSCs have been the most active for the treatment of ischemic myocardium. Cell transplantation has been shown to improve heart rate and cardiac function of the ischemic heart in patients with acute or chronic myocardial infarction (Stamm, C., et al., 2003, Lancet 361 : 45-46 .; Strauer, BE, et al., 2002, Circulation 106 : 1913-1918 .; Strauer, BE, et al., 2005, J. Am . Coll . Cardiol . 46 : 1651-1658; Tse, HF, et al., 2003, Lancet 361 : 47-49 Yamada, Y., et al., 2007, Stem. Cells 25 : 1326-1333 .; Yoo, KJ, et al., Can . J. Surg . (in press). Reference).

ADSCs include self-renewing stem cells capable of differentiating into osteogenic, adiogenic, myogenic, and chondrogenic lineages in vitro when treated with established lineage specific factors (Huang, JI). , et al, 2004, Plast Reconstr Surg 113:..... see 4279-4295):.... 585-594 .; Zuk PA, et al, 2002, Mol Biol Cell 13. ADSCs are easier to extract from patients than other types of stem cells and have the advantage of being able to extract clinically appropriate amounts of stem cells (Helder, MN, et al., 2007, Tissue Eng . 13 : 1799-). 1808.). In particular, transplantation of stem cells after differentiation into cardiomyocytes results in better cardiomyocyte regeneration and higher cardiac function improvement than transplanting undifferentiated stem cells (Li, TS, et al., Supra). In addition, undifferentiated stem cell transplantation can undergo differentiation into cells of other organs that are unexpected.

Several methods of differentiating stem cells into cardiomyocytes are known. Co-culture of BMMSCs and cardiomyocytes induces BMMSCs to differentiate into cardiomyocytes (Fukuhara, S., et al., 2003, J. Thorac . Cardiovasc . Surg . 125 : 1470-1480 . ; Rangappa, S., et al., 2003, Ann . Thorac . Surg . 75 : 775-779. Treatment with 5-azacytidine results in BMMSCs (see Makino, S., et al., 1999, 103 : 697-705.) And ADSCS (Rangappa, S., et al., 2003, Ann . Thorac . Surg 75:.. 775-779 reference) the obtained myocardial cell phenotype. Transformation growth factor-β1 (TGF-β1) and TGF-β2 are in vitro BMMSCs (Li, TS, et al., Supra) and embryonic stem cells (Kuma, D., et al., 2005, Biochem .... Biophys Res Commun 332 :. see 135-141) were stimulated to differentiate into cardiomyocytes. Bone forming protein-2 in rat embryonic stem cells also promotes cardiac gene expression (see Menard, C., et al., 2005, Lancet 366 : 1005-1012.).

Recently, stem cells have been identified in adipose tissue cells. These cells are called adipose derived stromal cells or adipose derived stem cells. ADSCs, like other mesenchymal stem cells, express multiple CD cell surface marker antigens such as CD29, CD44, CD90, and D105 (Gronthos, S., et al., 2001, J. Cell . Physiol . . 189:.. 54-63, Miyahara , Y, et al, 2006, Nat Med 12:.. 459-465 reference). Multipotents of ADSCs have been demonstrated for their ability to differentiate into various lineages including adipocytes, osteoblasts, chondrocytes, and endothelial cells (Afizah, H., et al., 2007, Tissue Eng . 13 : 659-666 .; Cao, Y, et al., 2005, Biochem . Biophys . Res . Commun . 332 : 370-379 .; Halvorsen, YD, et al., 2001, Tissue Eng . 7 : 729-741; Zuk PA, et al., 2001, Tissue Eng . 7 : 211-228 .; Zuk PA, et al., 2002, Mol . Biol . c ell . 13 : 4279-4295. Reference). Contractile cells with cardiomyocyte characteristics due to spontaneous differentiation appear during the culture of fat stromal cells, but their frequency is very low (0.02% to 0.07%, Cho, SW, et al., Eur . J. Heart Fail . (in press)).

Recent studies have co-cultured with cardiomyocytes (Fukuhara, S., et al., 2003, J. Thorac. Cardiovasc . Surg . 125 : 1470-1480 . ; Rangappa, S., et al., 2003, Ann . Thorac . Surg 75:.. 775-779 reference) 5 - if tidin processing between aza (Makino, S., et al, 1999, 103:.. 697-705 reference) reported that can be induced to differentiate into cardiomyocytes BMMSC It was. However, the co-culture method shows low cardiomyocyte differentiation (only 1.9%), and because 5-azacytidine is potentially toxic and has nonspecific dimethylation activity (Liu, Y., et al., 2003, Res . 58 : 460-468.) These methods suggest that they will not be clinically useful.

TGF-β1 is known to play an important role in embryonic cardiac development (see Akhurst, RJ, et al., 1990, Development 108 : 645-56.). TGF-β1 has been shown to induce differentiation into cardiomyocytes in mouse ES cells (Behfar, A., et al., 2002, FASEB J. 16 : 1558-1566 .; Kuma, D., et al., 2005 , Biochem Biophys Res Commun 332:. ... see 135-141). TGF-β1 treated CD117-positive BMMSCs have increased expression of cardiomyocyte markers such as Troponin-I, Troponin-T, Connexin-43, GATA-4, and NK _X -2.5 (Li, TS, et. al., supra).

On the other hand, TGF-β1 can induce osteogenic differentiation of BMMSC (Moses, HL, et al., 1996, Curr . Opin . Genet . Dev . 6 : 581-86 .; Li, TS, et al., remind). Recent studies have shown that CD117-positive BMMSCs incubated with low concentrations of TGF-β1 do not increase alkaline phosphatase activity in vitro and that high concentrations of TGF-β1 induce osteoblast differentiation (Li, TSet al). .remind). In addition, one study reported that TGF-β1 upregulates the expression of early striated myocyte differentiation markers such as SM α-actin and SM 22a in non-striated myocytes (Hautmann, MB, et al., 1999, Arterioscler. Thromb . Vasc . Biol . 19 : 2049-2058.).

Thus, a simpler, less toxic and clinically applicable cell for treating myocardial infarction that does not have the problem of transplanting conventional undifferentiated stem cells but does not have the above-mentioned problems from transplantation of known differentiated cells. The development of new culture and cell differentiation methods is urgently needed.

We have found the possibility of using adipose derived stromal cells (ADSCs) as a source of differentiated cardiomyocytes that can be used for transplantation. Surprisingly, the inventors have found that ADSCs differentiate into cardiomyocytes at a practical level when TGF is treated and cultured in vitro for a period of time, thus completing the present invention.

Reverse transcriptase polymerase reaction (RT-PCT), immunocytochemistry, and flow cytometry analysis indicated that TGF-β1 treatment, a type of TGF, augments the expression of various myocardial specific labels. The differentiation was also enhanced by increasing the duration of TGF-β1 treatment from 1 week to 2 weeks.

That is, cells differentiated according to the culture method of the present invention expressed cardiac α-actin, troponin-I, cardiac MHC, and α-sarcomeric actin (FIGS. 2-5). The proportion of ADSCs expressing cardiac MHC is very high (17%, FIG. 5).

In addition, ADSCs incubated with TGF-β1 at 10 ng / mL for 2 weeks did not express osteospecific tin or osteopontin, which are bone specific markers.

Accordingly, one aspect of the present invention provides a method for culturing fat-derived stromal cells comprising adding TGF to adipocyte-derived stromal cells in vitro to differentiate them into cardiomyocytes. The amount of TGF used to promote cardiomyocyte differentiation is preferably 1-100 ng / ml. More preferably, the amount of TGF used is about 10 ng / ml.

The TGF may be any kind of TGF capable of differentiating adipose derived stromal cells into an effective ratio of cardiomyocytes. Preferably the TGF is TGF-β1.

As used herein, 'Adipose-derived Stromal Cells (ADSCs)' or 'Adipose-derived Stem Cells' refer to cells having a multipotency identified in adipose tissue cells. ADSCs express a variety of CD cell surface labeled antigens, similar to other mesenchymal stem cells. Has the ability to differentiate into various lineages including adipocytes, osteoblasts, chondrocytes, and endothelial cells. Therefore, all culture methods for inducing cardiomyocyte differentiation similar to the method of the present invention using adipose derived stem cells are within the scope of the present invention.

In the above culturing method, the period of culturing fat-derived stromal cells by adding TGF is preferably 2 days or more. It will be apparent to those skilled in the art that the treatment duration may be increased or decreased depending on the differentiation inducing properties of the added TGF. The treatment period for inducing differentiation may select a period for inducing optimal differentiation by observing differentiation induction results.

Another aspect of the invention provides a myocardial cell population derived from adipose derived stromal cells differentiated into cardiomyocytes by culturing with the addition of TGF to the adipose derived stromal cells in vitro.

In another aspect of the present invention, there is provided a mammalian allograft or xenograft cell composition comprising the cardiomyocyte cell population according to the present invention. The mammal is preferably a human.

The culture method and the myocardial cell population according to the present invention increase the practicality of ADSCs as a transplant cell therapy that can be used for the treatment of myocardial infarction, and are useful for the production of such therapeutic agents. Pre-differentiation of ADSCs into cardiomyocytes using this method prior to transplantation is useful for improving the effect of treating myocardial infarction.

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

<Example>

Materials and methods

Cell separation

ADSC isolation and culture were described in Miyahara, Y, et al., 2006, Nat . Med . 12 : 459-465. Adipose tissue was isolated from the inguinal part of Sprague-Dawley rats (200-250 g, SLC, Tokyo, Japan). The isolated adipose tissue was rinsed in sterile phosphate buffered drinking water (PBS; Sigma, St. Louis, MO, USA) to remove contaminants, chopped with scissors, and then type 1 collagenase solution (0.1 mg / ml, Sigma) was treated with 10 ml at 37 ° C. for 1 hour. Filter with a 40 μm mesh filter (Falcon, Bedford, Mass.), And after centrifugation, the isolated cells were treated with 10% (v / v) fetal calf serum (FBS, Gibco BRL), 100 U / mL penicillin (Gibo BRL), and 0.1 mg / mL streptomycin (GiBco BRL) was added to α-MEM (α-minimal essential medium, Gibco BRL, Gaithersburg, MD, USA) incubated at 37 ° C. with 5% CO ₂ dispersed in air. Cells were secondary cultured at 60% confluency. To induce cardiomyocyte differentiation, cells were incubated for 2 weeks with the addition of 10 ng / mL TGF-β1 (R & D systems, Minneapolis, MN, USA). ADSCs not treated with TGF-β1 were used as controls. To induce adipose differentiation, confluent ADSCs were prepared using 3% (v / v) FBS, biotin (33 μmol / l), pantothenic acid (17 μmol / l), human recombinant insulin (1 μmol / l), Incubation was performed in DMEM-Ham's F-12 medium to which dexamethasone (1 μmol / l), isobutylmethylgxanthine (0.1875 mmol / l) and indomethic acid (0.2 mmol / l) were added. After 3 days induction phase, the cells were incubated in the same medium without isobutylmethylxanthine and the rest.

Flow cytometry analysis

For surface antigen phenotype, 2% (v / v) FBS containing mouse IgG and IgM or fluorescein isodiocyanic acid (FITC)-or phycoerythrin (PE) -binding antibody quantification after rinsing ADSCs with PBBS Incubated in / PBS buffer for 30 minutes at 4 ℃. Antibodies were CD34-PE, CD-37-PE, CD90-PE (BD Pharmingen, Los Angeles, CA, USA), and CD106-FITC (CHEMICON, Temecula, CA, SA). Mouse IgM-FITC (CHEMICON), mouse IgG1-PE, IgM-PE, rat IgG2B-PE, and mouse IgG-APC (BD Pharmingen) were used as isotype controls. ADSCs incubated with TGF-β1 were measured by flow cytometry analysis. After rinsing cells with PBS, incubate in 2% (v / v) FBS / PBS buffer for 5 minutes, incubate in 0.01% sodium azide (Sigma) PBS for 20 minutes, stain with anti-cardiac MHC, and then Treatment with FITC-binding anti-mouse IgG antibody. The samples were analyzed by Vantage SE flow cytometer (Bectondickinson, Franklin Lakes, NJ, USA) using CellQusest software (Becton Dickinson).

RT - PCR

RT-PCR analysis was performed in Hong, KH, et al., 2005, J. Microbiol . Biotechnol . 15 : 823-830. All RNA was isolated from wild rat heart, ADSCs, and osteoblasts using TRIzol reagent (Invitrogen, Carlsbad, Ca, USA). The specimen was dissolved in 200 μl of chloroform, homogenized with 1 mL of TRI zol reagent, and centrifuged at 12,000 rpm for 15 minutes. RNA pellets were rinsed with 70% (v / v) ethanol, dried and dissolved in water without RNase. Reverse transcription was performed with 5 μl of pure RNA using SuperScript ^™ II reverse transcriptase (Invitrogen). Synthesized cDNA was amplified by PCR using the primers shown in Table 1 below. PCR was performed by denaturation (94 ° C., 30 sec), annealing (60 ° C., 30 sec), and extension (72 ° C., 60 sec), last extended 30 times at 72 ° C. for 5 min. PCR products were visualized by electrophoresis on agarose gels with 2% (w / v) ethidium bromide staining and analyzed by gel documentation system (Gel Doc 1000, Bio-Red Laboratories, Hercules, Calif., USA). GAPDH was used as a "housekeeping" mRNA control.

Target order references Cardiac α-actin S: 5'-ACTCTATGTGTAGGTGACGAGGC-3 'AS: 5'-GACGTTATGAGTCACACCGTCG-3' Zwadlo C. et al., Supra Troponin I S: 5'-ATCTCCGCCTCCAGAAAACT-3 'AS: 5'-CAGTAGTGCCTGCATCATGG-3' Zwadlo C. et al., Supra Osteonectin S: 5'-GTG CCG AGA GTT CCC AGC ATC ATG-3 'AS: 5'-ATT CCT CCA GGG CAA TGT ACT TGT-3' Park, M.S. et al., Supra Osteocalin S: 5'-ATG TGC CCT CCT GGT TCA TTT CTT-3 'AS: 5'-GTG GTC CGC TAG CTC GTC ACA ATT-3' Park, M.S. et al., Supra GAPDH S: 5'-CCTCCTCATTGACCTCAACTAC-3 'AS: 5'-CATGGTGGTGAAGACGCCAG-3' Zhang F.B. et al., Supra

Immunofluorescence staining

Immunofluorescence staining was performed using Kim, H.C. et al., 2006, J. Microbiol. It was performed as described in Biotechnol. In summary, ADSCs were fixed in 2% paraformaldehyde (Sigme) for 15 minutes at room temperature. Cyanine-linked anti-goat IgG or second anti-mouse IgG antibody after staining ADSCs with antibody anti-cardiac MHC (Santa Cruz Biotechnology Inc.) and eradicated α-actinine (Sigma, St. Louis, MO, USA) (Jackson ImmunoResearch Laboratories). ADSCs were also stained with antibodies against smooth muscle (SM) α-actin (DAKO, Carpinteria, Calif.) And treated with FITC-binding anti-mouse IgG (Jackson ImmunoResearch Laboratories). The immunofluorescence staining samples were analyzed using confocal microscopy (LSM510, Carl Zeiss, Oberkochen, Germany). Cell nuclei were stained using Vectashield mounting medium with 4-6-diamino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, Calif., USA).

Alkaline  Phosphatase and oil red -O dye

Rat ADSCs and adipocytes were fixed in 10% (v / v) formaldehyde buffer for 1 hour and stained for 10 minutes in distilled water with 60% (v / v) oil red-O (Sigma) solution. Cells were then washed with distilled water, removed with residual oil-red-O solution and counterstained with hematoxylin. In order to determine the osteotyping, rat ADSCs and osteoblasts were stained for ALP with a commercial assay kit (Sigma) (Song, HB, et al., 2006, J. Microbiol. Biotechnol . 16 : 37-45. Reference).

Example  1: derived from fat Stromal cells  Property check

Based on the adherence of the cells, ADSCs were isolated from adipose tissue of rats and measured via flow cytometry for normal stem cell surface markers (Figure 1). After the first passage, many ADSCs expressed CD90 and a few expressed CD105, which are mesenchymal cell surface markers. In contrast, the majority of cells are negative for CD34, hematopoietic or hemangioblast surface markers, and CD73, a hepatocellular cell surface marker. These results are similar to previous studies (Cao, Y, et al., 2005, Biochem . Biophys . Res . Commun . 332 : 370-379 .; Gronthos, S., et al., 2001, J. Cell . Physiol .. 189:. 54-63 .; Lee, RH, et al, 2004, Cell Physiol . Biochem . 14 : 311-24; Miyahara, Y, et al., 2006, Nat . Med . 12 : 459-465. Reference).

Example  2: derived from fat Stromal cells Into cardiomyocytes  Differentiation induction

( Example  2-1) derived from fat Stromal cells Cardiomyocytes  Induction of differentiation and its confirmation

TGF-β1 treated ADSCs showed cardiac specific gene expression not present in TGF-β1 untreated ADSCs. RT-PCR analysis showed expression of several cardiac genes, including cardiac α-actin and troponin-I, in ADSCs cultured with TGF-β1 but not in ADSCs cultured without TGF-β1. (FIG. 2). Troponin-I expression was observed after TGF-β1 treatment in ADSCs and incubation for 2 weeks.

Immunofluorescence staining and flow cytometry showed cardiac specific marker expression in TGF-β1 treated ADSCs. Some of the ADSCs cultured for 2 weeks with TGF-β1 treatment stained positive for cardiac specific markers, cardiac MHC and α-sarcomeric actin (FIGS. 3C, 4C). ADSCs cultured without TGF-β1 or ADSCs cultured for only one week with TGF-β1 were not stained for cardiac markers (Figures 3A, B, 4A, B). Flow cytometry analysis shows that the rate of cardiac MHC-positive cells increases with time incubated with TGF-β1 and that the rate of cardiac MHC-positive cells is negligible in ADSCs not treated with TGF-β1. (FIG. 5). When cultured with TGF-β1, ADSCs expressing cardiac MHC increased 73-fold.

( Example  2-2) Confirmation of Differentiation of Adipose-derived Stromal Cells into Adipose Cells

To determine whether culturing with TGF-β1 differentiates ADSCs into adipocytes, the cultured ADSCs were stained with oil red-O, an adipocyte specific stain. ADSCs treated with or without TGF-β1 were not stained by oil red-O (FIGS. 6A, B, C). In contrast, ADSCs cultured with adipocyte differentiation medium showed lipid droplets in adipocytes and stained with oil red-O (FIG. 6D).

( Example  2-3) Differentiation of Adipose-derived Stromal Cells into Bone Cells

To determine whether incubation with TGF-β1 induced ADSCs to differentiate into osteoblasts, ALP products and osteoblast-specific gene expression were compared with osteoblasts in ADSCs cultured with TGF-β1. ADSCs cultured with TGF-β1 were not stained for ALP, while osteoblasts were stained positive for ALP (FIGS. 7A-D). RT-PCR analysis showed that ADSCs cultured with TGF-β1 did not express osteonectin or osteocalcin (FIG. 7E), which also did not differentiate bone formation under these culture conditions. Imply no.

1 is a graph showing the flow cytometry of the immune phenotype of ADSCs isolated from rat adipose tissue and cultured after the first passage. Cell surface antigen (green or red is second FITC- or PE-binding antibody, respectively)

Figure 2 is an electrophoresis picture showing the results of RT-PCR analysis for cardiac specific gene expression of ADGFs not treated with TGF-β1 or treated with TGF-β1. GAPDH was used as a housekeeping mRNA control. M: 100 bp DNA molecular weight marker, lane 1: ADSCs cultured without TGF-β1 treatment, lane 2: ADSCs cultured with TGF-β1 treatment for 1 week, lane 3: ADSCs cultured for 2 weeks with TGF-β1 treatment , Lane 4: rat heart.

Figure 3 is a micrograph showing the results of cardiac MHC immunocytochemical staining in ADSCs that were not treated with TGF-β1 (A) or treated with TGF-β1 for 1 week (B) or 2 weeks (C). Red indicates cardiac MHC-positive cells and blue indicates cell nuclei. The ruler is 100 μm.

4 shows immunocytochemistry for α-sarcomeric actin in ADSCs cultured without treatment of TGF-β1 (A) or treated with TGF-β1 for 1 week (B) or 2 weeks (C). It is a micrograph showing the staining result. Green indicates α-sarcomeric actin-positive cells and blue indicates cell nuclei. Ruler indicates 100 μm.

5 is a flow cytometry diagram for cardiac MHC expression in ADSCs cultured without treatment of TGF-β1 (A) or treated with TGF-β1 for one week (B) or for two weeks (C). to be. Assay lines represent isotype controls. The data represent the percentage of cardiac MHC-positive cells with mean ± standard deviation.

6 shows oil red-O of ADSCs and rat adipocytes (D) cultured without treatment of TGF-β1 (A) or treated with TGF-β1 for 1 week (B) or 2 weeks (C). It is a micrograph showing the staining result. (A <B <C) ADSCs were not stained by oil red-O. (D) Adipocytes containing fat droplets in the cytoplasm were stained positively by Oil Red-O. Ruler indicates 100 μm.

7 shows ALP staining results of ADSCs and rat osteoblasts (D) cultured without treatment of TGF-β1 (A) or treated with TGF-β1 for 1 week (B) or 2 weeks (C). It is a micrograph showing. Only rat osteoblasts were stained positive for ALP. Ruler indicates 200 μm. Figure 7 (E) is an electrophoresis picture showing the results of PR-PCR analysis of bone formation gene expression in ADSCs cultured with TGF-β1 for 1 or 2 weeks or without TGF-β1 treatment. GAPDH was used as a housekeeping mRNA control. Lane 1: ADSCs cultured without TGF-β1 treatment. Lane 2: ADSCs cultured with TGF-β1 for 1 week. Lane 3: ADSCs incubated with TGF-β1 treatment for 2 weeks. Lane 4: rat osteoblasts.

Claims (6)

A fat-derived stromal cell culture method comprising the step of adding TGF-β1 that promotes myocardial differentiation to adipocyte-derived stromal cells in vitro to differentiate them into cardiomyocytes. The method of culturing fat-derived stromal cells according to claim 1, wherein the amount of TGF-β1 is 1-100 ng / ml. The culture method according to claim 1 or 2, wherein the period of culturing with the addition of TGF-β1 is 2 days or more. delete delete delete

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