저작자표시-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게
l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. l 이 저작물을 영리 목적으로 이용할 수 있습니다.
다음과 같은 조건을 따라야 합니다:
l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다.
l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다.
저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다.
Disclaimer
저작자표시. 귀하는 원저작자를 표시하여야 합니다.
변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.
약학석사학위논문
Peptide hydrogel for stem cell delivery
줄기세포 전달을 위한 펩타이드 하이드로젤
2015 년 2 월
서울대학교 융합과학기술대학원 분자의학 및 바이오제약학과
김 건 우
Peptide hydrogel for stem cell delivery
줄기세포 전달을 위한 펩타이드 하이드로젤
지도교수 이 동 수
이 논문을 약학석사학위논문으로 제출함
2014 년 12 월
서울대학교 융합과학기술대학원
분자의학 및 바이오제약학과 김 건 우
김건우의 석사학위논문을 인준함 2014 년 12 월
위 원 장 변 영 로 ( 인 )
부 위 원 장 이 동 수 ( 인 )
위 원 오 유 경 ( 인 )
Abstract
Peptide hydrogel for stem cell delivery
Gunwoo Kim, Molecular Medicine and Biopharmaceutical Sciences, WCU Graduate School of Convergence Science and Technology, Seoul National University, Seoul National University
Here, we report biomimetic chimeric peptide-tethered bioactive fibrin hydrogel scaffold for enhanced survival of human mesenchymal stem cells (hMSC). We designed biomimetic chimeric peptide to tether and stimulate hMSC, and to anchor hMSC to fibrin gel matrix. As an hMSC-tethering moiety, two types of peptides were compared for cell adhesion ability. RGD and osteopontin-derived peptide (OP) showed similar adhesion to hMSC. However, hMSC-proliferation ability was observed in SVV, but not in RGD. To anchor hMSC to fibrin gel, natural fibrin-binding protein-derived peptide (FBP) was tested. The loading of FBP to fibrin gel was 8.2-fold higher as compared to that of scrambled peptide of same amino compositions of FBP (scFBP).
Fluorescent FBP-loaded fibrin gels retained at the injection site longer than fluorescent scFBP-loaded fibrin gels. At 48 hr after injection, the photon intensity of fluorescent FBP-loaded fibrin gels was 15.9-fold higher than that of fluorescent scFBP-grafted fibrin gels. Given the fibrin gel-binding properties of FBP and the hMSC-binding and proliferation properties of OP, chimeric peptides were constructed to have FBP and OP using a spacer linker (FBPsOP). Four days after transplantation, the number of residual hMSC in FBPsOP-grafted fibrin gels was 3.9-fold higher as compared to hMSC in fibrin gel alone.
Our results suggest the potential of the FBPsOP-grafted fibrin gel as a bioactive scaffold for transplantation of stem cells.
Key words: biomimetic chimeric peptide, fibrin binding peptide, osteopontin-derived peptide, human mesenchymal stem cells, cell survival.
Student number : 2013-22722
Contents
Ⅰ . Introduction
Ⅱ . Materials and methods
2. 1. Culture of hMSC...3
2. 2. Peptides...4
2. 3. In vitro hMSC adhesion and proliferation test...4
2. 4. Animals...5
2. 5. Measurement of FBP binding to fibrin gel...5
2. 6. Molecular imaging of fluorescent FBP-loaded fibrin gel...6
2. 7. Measurement of hMSC after transplantation...6
Ⅲ . Results
3. 1. Identification of hMSC...8
3. 2. Adhesion and proliferation effect of peptides...10
3. 3. Binding of FBP to fibrin gel matrix ...13
3. 4. In vivo survival of hMSC transplanted with
FBPsOP - grafted fibrin gels...15
Ⅳ . Discussion
Ⅴ . Conclusion
Ⅵ . References
국문초록
Lists of Figures
Figure 1. Characterization of hMSC.
Figure 2. Cell adhesion of OP on hMSC.
Figure 3. Proliferation effect of OP on hMSC.
Figure 4. Fibrin gel binding affinity of F-FBP.
Figure 5. Survival of transplanted hMSC in
FBPsOP-grafted fibrin gel.
Ⅰ. Introduction
Stem cell-based therapeutics has been in the spotlight due to the promising potentials including multiple lineage differentiation, self-renewal ability, and immune suppression [Naji et al., 2013; Lo et al., 2013]. Despite hundreds of stem cell therapeutics are undergoing clinical trials, there still are several obstacles to overcome in the field of cell-based medicine [Bianco et al., 2013; Sharma et al., 2014]. One obstacle is low survival rate of stem cells after transplantation [Das et al., 2010; Huang et al., 2014]. For this reason, survival enhancers which can maintain survival of co-transplanted cells have been studied as antidotes for therapeutic use of stem cells [Shim et al., 2013; Wang et al., 2014].
Hydrogels have been widely used in medical field for tissue implantation because it can mimic the natural extracellular environment with three dimensional networks and provide stable settlement of transplanted cells [Seliktar, 2012; Aguado et al., 2012]. Recently, application of bioactive substances to hydrogel matrix has received much attention. Hydrogels have been conjugated or physically encapsulated with bioactive substances such as growth factors, peptides, and small molecules which can improve survival and modulate differentiation of stem cells [Guvendiren and Burdick, 2013; Jeon et al., 2013; Ziv et al., 2014]. However, such physical encapsulation or chemical conjugation still suffers from limitations. Physical encapsulation lacking anchoring moieties to hydrogel matrix could cause quick release of bioactive adjuvant from hydrogel. Meanwhile, chemical conjugation of bioactive substance to hydrogels requires complicated synthetic process and can reduce
the biological functions after conjugation [Park et al., 2014; Egusa et al., 2009].
Here, to enhance the stem cell survival after transplantation, we designed a hydrogel grafted with chimeric peptide composed of hydrogel matrix anchoring moiety and stem cell adhering moiety. As a hydrogel matrix anchoring moiety, we took advantage of natural protein and protein-binding peptide systems. Fibrin is known to bind with fibrin-binding peptide [Sacchi et al., 2014]. As a stem cell tethering moiety, we used osteopontin-derived peptide (OP). In this study, we tested whether the survival of human bone marrow-derived mesenchymal stem cells (hMSC) could be enhanced following transplantation in the chimeric peptide-grafted fibrin hydrogels.
Ⅱ. Materials & Methods
2. 1. Culture of hMSC
Bone marrow aspirates were obtained from a healthy male donor after informed consent (approved by the INHA University Medical School Institutional Review Board; IRB Number 10-51). Isolation of hMSC was performed as previously described [Yi et al. 2014]. Cells were maintained in Dulbecco’s modified Eagle medium (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco), 100 units/ml of penicillin, 100 mg/ml of streptomycin, and 25 mg/ml of amphotericin B at 37
°C in a humidified atmosphere containing 5% CO2. The non-adherent cells were discarded and the adherent cells were cultured to confluence with medium changes every 3 day. Expanded hMSC were harvested using a 0.25
% trypsin/ethylenediamine tetraacetic acid solution (Gibco) after washing twice with phosphate-buffered saline (PBS; pH 7.4). Cell pellets were collected by centrifugation at 800 rpm for 3 min at 20 °C, and resuspended in culture media. Passage 9-12 hMSC were used in this study. The identity of cells was confirmed by analyzing hMSC markers using flow cytometry (BD Biosiences, Franklin Lakes, NJ, USA). For evaluation of differentiation potential, three mesenchymal cell types were independently induced and assessed by cell type-specific cytostaining. Adipogenic, osteogenic, and chondrogenic differentiation was performed as described [Yi et al., 2014].
2.2. Peptides
All peptides used in this study were purchased from Peptron (Daejeon, Korea). The sequences for RGD peptides and osteopontin-derived peptide (OP) were GRGDS and GRGDSVVYGLR, respectively. The amino acid sequence of fluorescein isothiocyanate-labeled fibrin gel binding peptide (F-FBP) was NQEQVSP-GGGSK-FITC. The sequence of fluorescein isothiocyanate-labeled scrambled FBP (F-scFBP) was PQSQENV-GGGSK-FITC. Chimeric peptide of FBP and OP was NQEQVSP-GGGS-GRGDSVVYGLR, in which GGGS was introduced as a spacer.
2.3. In vitro hMSC adhesion and proliferation test
To test the adhesion property of peptides to hMSC, hMSC was seeded onto peptide-bound plate, and the adhesion extent of hMSC was quantitated by testing the viability of cells on peptide-bound plates. To generate peptide-bound plate, RGD or OP was incubated in amine binding plate (BD PureCoat, BD Biosiences) for 12 h at 4°C. Then, the plates were washed twice with PBS to remove unbound peptides, and seeded with hMSC at a density of 1ｘ104 cells per well. After 4 h incubation, the adhesion extent of hMSC onto peptide-coated plates was evaluated using a 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-aldrich, St. Louis, MO, USA). MTT solution (500 mM) was added and plate was incubated for an additional 1 h. The resulting crystal were dissoleved in 100 ml of 0.06 N HCl in isopropanol, and the absorbance was measured at 570 nm using a microplate reader (Sunrise Basic; TECAM,
Männedorf, Switzerland).
For cell proliferation test, hMSC were seeded in 24 well-plates at a dose of 1.5ｘ104 cells per well, and maintained in media containing 2 mM of RGD or OP. The cells were cultured with replacement of culture medium containing 2 mM of peptide every other day. The cell viability was evaluated using MTT assay.
2.4. Animals
All in vivo experiments were tested using six-week-old female Balb/c athymic nude mice supplied from Orient Bio. Lab. Animal Inc. (Seungnam, Kyonggi-do, Republic of Korea, approved animal experimental protocol number SNU-130129-3-1). Animals were raised under standard pathogen-free conditions at the animal center for pharmaceutical research in Seoul National University. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, Seoul National University.
2.5. Measurement of FBP binding to fibrin gel
Fibrin gel was formed using a commercially available, thrombin-induced fibrin gel kit (Greenplast; Green Cross Co., Gyeonggi-do, Korea). Fifty micro liter of 0.4 mM thrombin solution was premixed with 0.12 mM of F-FBP or F-scFBP peptides and added to 50 ml of 0.03 mM fibrinogen solution at a molar ratio of 4:1 (peptide: fibrinogen). After gel formation, gels were placed in a dialysis bag (Sectrum Laboratories, Inc., Rancho Dominguez, CA, USA) against PBS at 37 °C. After 24 h, 0.25 % trypsin-EDTA solution was used
to dissolve gels and fluorescence intensity of FITC-labeled peptides bound in the fibrin gel was measured at 525 nm using a fluorescence microplate reader (Gemini XS; Molecular Device, Sunnyvale, CA, USA).
2.6. Molecular imaging of fluorescent FBP-loaded fibrin gel
To test whether FBP was retained in the fibrin gel matrix in vivo, mice were subcutaneously transplanted with fluorescent FBP-loaded fibrin gels.
Fifty micro liter of thrombin solution was premixed with 0.12 mM of F-FBP or F-scFBP. After subcutaneous injection into mice, 50 ml of 0.03 mM fibrinogen solution was subsequently injected to induce gel formation. After 24 h, the transplanted gels were extracted and washed several times in PBS.
The fluorescence intensity of dissolved gel solution intensity was measured by the fluorescence microplate reader (Molecular Device).
2.7. Measurement of hMSC after transplantation
Mice were subcutaneously transplanted with 2.5 × 105 hMSC in a thrombin-induced fibringel containing OP or FBPsOP. After centrifugation, pellets of hMSC (2.5×105 cells) were dispersed with 50 ml of thrombin solution containing 0.12 mM of OP or FBPsOP. After subcutaneous injection into mice, 50 ml of 0.03 mM fibrinogen solution was subsequently injected to induce gel formation at a molar ratio of 4:1 (peptide: fibrinogen). After 4 day, the cells at the transplantation site were collected and gDNA was extracted and purified using a DNeasy Tissue Kit (Qiagen, Hilden, Germany), as described by the manufacturer. A hAlu element was selected as a marker of gDNA of transplanted hMSC for qRT-PCR. The primer (Macrogen, Seoul,
Korea) for hAlu were 5′-GCCTGTAATCCCAGCACTTT-3′ (sense) and 5′
-CACTACGCCCGGCTAATTT-3′ (antisense). After extraction of the gels and purification of gDNA from hMSC, Quantitative real-time PCR was performed in 20 ml glass capillaries using a LightCycler 2.0 instrument with LightCycler FastStart, DNA Master PLUS SYBR Green І reagents; data were analyzed using the LightCycler software program (Roche Diagnostics GmbH, Mannheim, Germany). Thermocycling parameters consisted of a hot start at 95 °C for 10 min followed by 30 cycles of 95 °C for 10 s, 57 °C for 20 s, and 72 °C for 20 s. A melting curve analysis was performed to confirm the specificity of the PCR products after the amplification step.
Ⅲ. Results
3.1 Identification of hMSC
The hMSC were identified by several stem cell makers and differentiation potentials (Fig. 1). The cells were positively stained with various hMSC markers (CD29, CD44, CD90, CD105, CD146, HLA-I, and OCT4) but hardly stained with hematopoietic/ endothelial markers (CD14, CD34, CD45, and HLA-DR) (Fig.1 A). Oil red O (Fig.1 B), alizarin red S (Fig.1 C), and safranin O (Fig.1 D) staining data revealed that adipogenic, osteogenic, and chondrogenic potentials of hMSC, respectively.
Figure 1. Characterization of hMSC
Expression of several stem cell markers was analyzed by flow cytometry (A).
Multi-lineage differentiation potential of hMSC was evaluated by cell type-specific cytostaining using oil red O (B), alizarin red S (C), and safranin O (D). Scale bar = 20 mm.
3.2. Adhesion and proliferation effect of peptides
The OP affected cellular adhesion (Fig. 2) and proliferation of hMSC (Fig. 3). The adhesion of hMSC onto OP- or RGD-coated plate was tested as illustrated in Fig. 2A. OP increased hMSC adhesion in a comparable manner to RGD. OP and RGD increased the adhesion of hMSC 1.27 ± 0.66-fold, and 1.17 ± 0.45-fold, as compared with peptide non-treated group, respectively (Fig. 2B). Although OP showed similar hMSC adhesion ability to RGD, OP but not RGD, significantly enhanced the proliferation of hMSC (Fig. 3). hMSCs were treated with OP or RGD as illustrated in Fig. 3A.
Proliferation of hMSC was measured as viability of hMSC using MTT assay.
Treatment of adhered hMSC with RGD did not affect the viability of hMSC over 4 days after treatment. However, OP treatment increased the viability of hMSC to 112.0 ± 1.5% and 116.9 ± 2.8% on days 2 and 4 as compared to that of untreated hMSC group (Fig. 3B).
Figure 2. Cell adhesion of OP on hMSC.
(A) RGD peptide or OP-coated plate was treated with hMSC for 4 h. (B) The adhesion of hMSC onto the peptide-coated plate was compared with the adhesion of hMSC on peptide-uncoated plate by MTT assay.
Figure 3. Proliferation effect of OP on hMSC.
(A) hMSCs were treated with RGD or OP peptide for 4 days. (B) The proliferation of hMSC was evaluated by MTT assay. The results are expressed as means ± SDs (n = 6). *Significantly higher (p < 0.05) compared to the untreated or RGD group on same time points (assessed by ANOVA and Student-Newman-Keuls test).
3.3. Binding of FBP to fibrin gel matrix
The schematic diagram of F-FBP-anchored fibrin gel is depicted in Fig.
4A. As shown in Fig. 4B, fibrin gel was formed with fibrinogen and thrombin solution which was premixed with F-scFBP or F-FBP. Scrambled peptide of same amino compositions of FBP (scFBP) has no fibrin binding ability and so, it was used for negative control. F-FBP-grafted fibrin gel showed the strong yellowish color of FITC fluorescence whereas F-scFBP-grafted fibrin gels showed little yellowish color of gel-retained FITC.
Fluorescence intensity of dissolved F-FBP gel was 8.2-fold higher than that of F-scFBP (Fig. 4C).
In addition to the in vitro study, binding affinity of FBP to fibrin gel was evaluated in vivo after subcutaneous injection of hydrogels to mice (Fig.
4D). F-FBP-grafted fibrin gels showed higher fluorescence over 48 h as compared with F-scFBP-grafted gels. The photon count of F-FBP-grafted fibrin gel-injection site was 15.9-fold higher than that of F-scFBP-grafted gel injection site at 48 h post-injection.
Figure 4. Fibrin gel binding affinity of F-FBP
(A) The structure of F-FBP grafted fibrin gel matrix was illustrated. (B) Thrombin-induced fibrin gels were formed containing FITC-labeled FBP or scFBP at a molar ratio of 4:1 (peptide to fibrinogen) and then dialyzed in PBS. After 24 h, the fibrin gels (B) were dissolved for fluorometry (C).
Fibrin gels containing F-FBP or scF-FBP (peptide: fibrinogen = 4:1, m/m) were extracted and analyzed by fluorometry at different time after transplantation of into mice. The results are expressed as means ± SDs (n = 4). *Significantly higher (p < 0.05) compared to the scF-FBP group (C) on same time point (D) (assessed by ANOVA and Student-Newman-Keuls test).
3.4 In vivo survival of hMSC transplanted with FBPsOP-grafted fibrin gels
Survival of transplanted hMSC was modulated by the grafting method of OP to fibrin gel (Fig. 5). At 4 days post-transplantation, OP-entrapped fibrin gel increased the survival of hMSC to 1.3-fold as compared with fibrin gel alone. In contrast, FBPsOP-grafted fibrin gel provided 3.9-fold higher survival of hMSC as compared to fibrin gel alone.
Figure 5. Survival of transplanted hMSC in FBPsOP-grafted fibrin gel
Mice were subcutaneously transplanted with hMSC in OP-entrapped fibrin gel or FBPsOP-grafted fibrin gel. After 4 days, the fibrin gels were collected, and the survival of hMSC was quantitated by qRT-PCR of extracted gDNA.
*Significantly higher (p < 0.05) compared to the other groups (assessed by ANOVA and Student-Newman-Keuls test).
Ⅳ. Discussion
In this study, we demonstrated that a biomimetic chimeric peptide composed of FBP and OP could be grafted to fibrin gels by natural binding mechanisms of fibrin and FBP without using chemical conjugation. In addition to the gel matrix-anchoring FBP, OP served as hMSC-tethering moiety. Moreover, OP could stimulate the proliferation of hMSC. We could achieve the higher survival of transplanted hMSC using FBPsOP-grafted fibrin gels.
Upon simple addition of FBP, FBP bound to fibrin gel by the natural specific binding mechanism, without chemical conjugation. The FBP motif, a domain from anti-plasmin a2, is recognized by factor XIII and binds to fibrin gels by enzymatic crosslinking [Padmashali et al., 2011; Riopel et al., 2014]. Recently, the application of FBP to protein functionalization was reported. In the study, FBP-engineered recombinant fragment of fibronectin could act as a binding moiety for growth factors cocktail [Martino et al., 2011]. Liang et al. constructed TGFb-1-immobilized fibrin gel by using FBP fusion protein which provided sustained signaling of growth factor in fibrin-embedded cells for several days [2011]. In terms of drug development, FBP chimeric peptide may have merits than FBP-fusion proteins. As compared to fusion protein, chimeric peptides has a much shorter size and easy to handle. Moreover, in the perspective of the manufacturing process, chimeric peptides can be easily produced in large scale by chemical synthesis without complex genetic engineering and purification processes of proteins.
The binding between FBP and fibrin gel was observed in vitro as well as in vivo (Fig. 4). The addition of bioactive substance to hydrogel matrix was studied to enhance the survival of stem cells [Egusa et al., 2009; Shim et al., 2013; Jung et al., 2014]. However, the approaches achieved a limited success due to the rapid leakage of bioactive substances from the matrix [Egusa et al., 2009]. To avoid chemical conjugation, streptavidin-biotin approaches have been studied to anchor stem cells to hydrogel matrix. [Li et al., 2014]. Our approach could eliminate the cumbersome additional modification of hydrogel matrix with external streptavidin.
We observed that OP increased the adhesion (Fig. 2) and proliferation (Fig. 3) of hMSC in vitro. OP, an oligopeptide originated from extracellular matrix protein osteopontin, is located next to RGD sequence in osteopontin [Egusa et al., 2009]. Consistent with our observation on the OP’s dual roles, several reports have presented multiple biological activity of OP including angiogenesis, cell proliferation, and binding ability to integrins [Uchinaka et al., 2013; Lei et al., 2012]. OP has been reported to bind to various cells such as endothelial cells [Lei et al., 2012; Park et al., 2014], and myoblasts [Uchinaka et al., 2013]. Although FBPsOP hydrogels were used to enhance the survival of hMSC in this study, the various cell adhesion properties of OP can make it possible for the FBPsOP hydrogels to be applied for delivery of other cell therapeutics.
We tested the retention of transplanted hMSC using qRT-PCR with hAlu as a marker (Fig. 6). We used hAlu primer for specific detection of hMSC since hAlu gene exists only in hMSCs and not in mouse cells. Previously, the in vivo trafficking of hMSC using hAlu was used to test the in vivo fate
of intraarticularly administered hMSC. The detection of hMSC with hAlu has merits to evaluate the survival in that we can avoid the extra processing of hMSC with fluorescent dyes or other detection markers.
We can increase retention time and survival rate of transplanted stem cell at the injected site, thus, we are able to enhance the stem cell therapeutic effect using chimeric peptide and fibrin gel. Also, using these chimeric peptide techniques can be applied to drug delivery system with various chimeric peptides (or drugs) and fibrin gel.
Ⅴ. Conclusion
In this study, we formulated hMSC in biomimetic FBPsOP chimeric peptide-grafted fibrin hydrogel. OP was used to adhere and stimulate the proliferation of hMSC. FBP was used to employ the natural specific and high affinity binding mechanisms of FBP and fibrin. We observed that FBP could bind to fibrin gel matrix in vitro and in vivo. The transplantation of hMSC in FBPsOP-grafted fibrin gel could retain at the injection sites with enhanced survival. Our result suggests the potential of FBPsOP-grafted fibrin hydrogels for delivery system of hMSC.
Ⅵ. References
1. Aguado BA, Mulyasasmita W, Su J, Lampe KJ, Heilshorn SC.
Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers. Tissue Eng Part A.
2012;18(7-8):806-815.
2. Bianco P, Cao X, Frenette PS, Mao JJ, Robey PG, Simmons PJ, Wang C-Y. The meaning, the sense and the significance:
translating the science of mesenchymal stem cells into medicine.
Nat Med. 2013;19(1):35-42.
3. Das R, Jahr H, van Osch GJ, Farrell E. The role of hypoxia in bone marrow-derived mesenchymal stem cells: considerations for regenerative medicine approaches. Tissue Eng Part B Rev.
2010;16(2):159-168.
4. Egusa H, Kaneda Y, Akashi Y, Hamada Y, Matsumoto T, Saeki M, Thakor DK, Tabata Y, Matsuura N, Yatani H.
Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts.
Biomaterials. 2009;30(27):4676-4686.
5. Guvendiren M, Burdick JA. Engineering synthetic hydrogel microenvironments to instruct stem cells. Curr Opin Biotechnol.
2013;24(5):841-846.
6. Huang B, Qian J, Ma J, Huang Z, Shen Y, Chen X, Sun A,
Ge J, Chen H. Myocardial transfection of hypoxia-inducible factor-1alpha and co-transplantation of mesenchymal stem cells enhance cardiac repair in rats with experimental myocardial infarction. Stem Cell Res Ther. 2014;5(1):22.
7. Jeon O, Alt DS, Linderman SW, Alsberg E. biochemical and physical signal gradients in hydrogels to control stem cell behavior. Adv Mater. 2013;25(44):6366-6372.
8. Jung O, Hanken H, Smeets R, Hartjen P, Friedrich RE, Schwab B, Gröbe A, Heiland M, Al-Dam A, Eichhorn W, Sehner S, Kolk A, Wöltje M, Stein JM. Osteogenic differentiation of mesenchymal stem cells in fibrin-hydroxyapatite matrix in a 3-dimensional mesh scaffold. in vivo.
2014;28(4):477-482.
9. Kim B-S, Kim H-J, Choi J-G, You H-K, Lee J. The effects of fibrinogen concentration on fibrin/atelocollagen composite gel: an in vitro and in vivo study in rabbit calvarial bone defect. Clin Oral Implants Res. 2014. doi: 10.1111/clr.12455. [Epub ahead of print]
10. Lei Y, Rémy M, Labrugère C, Durrieu M-C. Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration. J Mater Sci Mater Med. 2012;23(11):2761-2772.
11. Li H, Koenig AM, Sloan P, Leipzig ND. In vivo assessment
of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds. Biomaterials.
2014. doi: 10.1016/j.biomaterials.2014.07.038 [Epub ahead of print]
12. Liang MS, Andreadis ST. Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs. Biomaterials. 2011;32(33):8684-8693.
13. Lo WC, Chen WH, Lin TC, Hwang SM, Zeng R, Hsu WC, Chiang YM, Liu MC, Williams DF, Deng WP. Preferential therapy for osteoarthritis by cord blood MSCs through regulation of chondrogenic cytokines. Biomaterials. 2013;34(20):4739-4748.
14. Martino MM, Tortelli F, Mochizuki M, Traub S, Ben-David D, Kuhn GA, Muller R, Livne E, Eming SA, Hubbell JA.
Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing. Sci Transl Med. 2011;3(100):100ra189.
15. Naji A, Rouas-Freiss N, Durrbach A, Carosella ED, Sensébé L, Deschaseaux F. Concise review: Combining human leukocyte antigen G and mesenchymal stem cells for immunosuppressant biotherapy. Stem Cells. 2013;31(11):2296-2303.
16. Padmashali RM, Andreadis ST. Engineering fibrinogen-binding VSV-G envelope for spatially- and cell-controlled lentivirus delivery through fibrin hydrogels. Biomaterials.
2011;32(12):3330-3339.
17. Park KM, Lee Y, Son JY, Bae JW, Park KD. In situ SVVYGLR peptide conjugation into injectable gelatin-poly(ethylene glycol)-tyramine hydrogel via enzyme -mediated reaction for enhancement of endothelial cell activity and neo-vascularization. Bioconjug Chem. 2012;23(10):2042-2050.
18. Riopel M, Trinder M, Wang R. Fibrin, a scaffold material for islet transplantation and pancreatic endocrine tissue engineering.
Tissue Eng Part B Rev. 2014.
19. Sacchi V, Mittermayr R, Hartinger J, Martino MM, Lorentz KM, Wolbank S, Hofmann A, Largo RA, Marschall JS, Groppa E, Gianni-Barrera R, Ehrbar M, Hubbell JA, Redl H, Banfi A.
Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164. PNAS. 2014;111(19):6952-6957.
20. Seliktar D. Designing cell-compatible hydrogels for biomedical applications. Science. 2012;336(6085):1124-1128.
21. Sharma RR, Pollock K, Hubel A, McKenna D. Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices. Transfusion. 2014;54(5):1418-1437.
22. Shim G, Im S, Lee S, Park JY, Kim J, Jin H, Lee S, Im I, Kim D-D, Kim SW, Lee TJ, Eom JS, Yi TG, Song SU, Byun Y, Oh YK. Enhanced survival of transplanted human
adipose-derived stem cells by co-delivery with liposomal apoptosome inhibitor in fibrin gel matrix. Eur J Pharm Biopharm. 2013;85(3, Part A):673-681.
23. Snyder TN, Madhavan K, Intrator M, Dregalla RC, Park D. A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair. J Biol Eng.
2014;8:10.
24. Uchinaka A, Kawaguchi N, Hamada Y, Mori S, Miyagawa S, Saito A, Sawa Y, Matsuura N. Transplantation of myoblast sheets that secrete the novel peptide SVVYGLR improves cardiac function in failing hearts. Cardiovas Res.
2013;99(1):102-110.
25. Wang Y, Li C, Cheng K, Zhang R, Narsinh K, Li S, Li X, Qin X, Su T, Chen J, Cao F. Activation of liver X receptor improves viability of adipose-derived mesenchymal stem cells to attenuate myocardial ischemia injury through TLR4/NF-kappaB and Keap-1/Nrf-2 signaling pathways. Antioxid Redox Signal.
2014.
26. Yamaguchi Y, Shao Z, Sharif S, Du XY, Myles T, Merchant M, Harsh G, Glantz M, Recht L, Morser J, Leung LLK.
Thrombin-cleaved fragments of osteopontin are overexpressed in malignant glial tumors and provide a molecular niche with survival advantage. J Biol Chem. 2013;288(5):3097-3111.
27. Yi T, Lee HJ, Cho YK, Jeon MS, Song SU. Molecular characterization of neurally differentiated human bone marrow-derived clonal mesenchymal stem cells. Immune Netw.
2014;14(1):54-65.
28. Ziv K, Nuhn H, Ben-Haim Y, Sasportas LS, Kempen PJ, Niedringhaus TP, Hrynyk M, Sinclair R, Barron AE, Gambhir SS. A tunable silk–alginate hydrogel scaffold for stem cell culture and transplantation. Biomaterials. 2014;35(12):3736-3743.
초 록
줄기세포 전달을 위한 펩타이드 하이드로젤
김건우, 분자의학 및 바이오제약학과, 융합과학기술대학원, 서울대학교
현재 줄기세포를 이용한 난치병 및 다양한 질병 치료들이 시도 되 고 있다. 하지만 이러한 줄기세포 치료에서 가장 큰 어려움 중 하 나가 줄기세포의 생존율이 낮다는 점이다.
이 논문은 생체모방형 키메릭 펩타이드와 피브린젤을 이용하여 줄 기세포의 생존율을 높이는 방법을 소개하고 있다. 키메릭 펩타이드 는 오스테오폰틴 유래 펩타이드와 피브린에 붙는 펩타이드 서열의 연결로 구성되어 있다. 오스테오폰틴 유래 펩타이드는 줄기세포의
생장 속도를 높여주는 역할 뿐 아니라 줄기세포를 잡아주는 역할을 하고, 피브린에 붙는 펩타이드 서열은 피브린 젤에 키메릭 펩타이 드가 잘 붙게 하여 궁극적으로 이 키메릭 펩타이드와 피브린 젤을 이용하여 줄기세포의 생존율을 높여주는 역할을 한다.
오스테오폰틴 유래 펩타이드와 RGD 펩타이드 모두 비슷한 줄기세 포의 부착능을 보여줬지만, 오스테오폰틴 유래 펩타이드만이 줄기 세포 생장률을 높여주는 것을 확인하였다. 또한, 피브린에 붙는 펩 타이드 서열이 실제로 스크램블 펩타이드 서열에 비해 피브린젤에 8.2배 더 잘 붙는 것을 형광을 이용하여 확인하였다. 마지막으로 오 스테오폰틴 유래 펩타이드와 피브린 젤을 이용하여 쥐에 줄기세포 를 이식 후 줄기세포의 생존율이 3.9배나 증가된 것을 확인하였다.
이처럼 본 연구에서는 키메릭 펩타이드와 피브린 젤을 이용하여 줄 기세포의 이식 후 생존율을 높여 줄기세포 치료 효능을 증가시킬 수 있는 가능성을 보여준다.
Key words:
biomimetic chimeric peptide, fibrin binding peptide, osteopontin-derived peptide, human mesenchymal stem cells, cell survival.학 번 : 2013-22722
