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Behavior, PET and Histology in Novel Regimen of MPTP Marmoset Model of Parkinson's Disease for Long-Term Stem Cell Therapy.

Jun Won YUN ; Jae Bum AHN ; Euna KWON ; Jae Hun AHN ; Hyung Woo PARK ; Hwon HEO ; Jin Sung PARK ; Hyeonjin KIM ; Sun Ha PAEK ; Byeong Cheol KANG

Tissue Engineering and Regenerative Medicine.2016;13(1):100-109. doi:10.1007/s13770-015-0106-3

Stem cell technologies are particularly attractive in Parkinson's disease (PD) research although they occasionally need long-term treatment for anti-parkinsonian activity. Unfortunately, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) widely used as a model for PD has several limitations, including the risk of dose-dependent mortality and the difficulty of maintenance of PD symptoms during the whole experiment period. Therefore, we tested if our novel MPTP regimen protocol (2 mg/kg for 2 consecutive days and 1 mg/kg for next 3 consecutive days) can be maintained stable parkinsonism without mortality for long-term stem cell therapy. For this, we used small-bodied common marmoset monkeys (Callithrix jacchus) among several nonhuman primates showing high anatomical, functional, and behavioral similarities to humans. Along with no mortality, the behavioral changes involved in PD symptoms were maintained for 32 weeks. Also, the loss of jumping ability of the MPTP-treated marmosets in the Tower test was not recovered by 32 weeks. Positron emission tomography (PET) analysis revealed that remarkable decreases of bindings of ¹⁸F-FP-CIT were observed at the striatum of the brains of the marmosets received MPTP during the full period of the experiment for 32 weeks. In the substantia nigra of the marmosets, the loss of tyrosine hydroxylase (TH) immunoreactivity was also observed at 32 weeks following the MPTP treatment. In conclusion, our low-dose MPTP regimen protocol was found to be stable parkinsonism without mortality as evidenced by behavior, PET, and TH immunohistochemistry. This result will be useful for evaluation of possible long-term stem cell therapy for anti-parkinsonian activity.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine* ; Brain ; Callithrix* ; Haplorhini ; Humans ; Immunohistochemistry ; Models, Animal ; Mortality ; Parkinson Disease* ; Parkinsonian Disorders ; Positron-Emission Tomography ; Primates ; Stem Cells* ; Substantia Nigra ; Tyrosine 3-Monooxygenase

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STX0119 Ameliorates Arthritis in SKG Mice via Inhibiting T Helper 17.

Faisal HAYAT ; Seung Hoon LEE ; Eun Jung LEE ; Seok Jung KIM ; KyungAh JUNG ; Soon Kyu LEE ; Jeehee YOUN ; Jun Ki MIN ; Mi La CHO ; Dong Yun SHIN

Tissue Engineering and Regenerative Medicine.2016;13(1):91-99. doi:10.1007/s13770-016-9086-0

Rheumatoid arthritis (RA) is an autoimmune disease with chronic and excessive inflammation. Upregulation of interleukin (IL)-17 is involved in the pathogenesis of RA. STX0119 is a specific inhibitor of signal transducer and activator of transcription 3 (STAT3) as a potential target for the treatment of RA. STAT3 is a member of DNA-binding molecules that regulates the expression of proinflammatory cytokines involved in the pathogenesis of RA. The objective of this study was to determine whether STX0119 could inhibit STAT3 and IL-17. We demonstrated that STX0119 decreased T helper (Th) 17 differentiation and IL-17 expression in vitro. STX0119 also improved the severity of zymosan induced arthritis and reduced joint inflammation. STX0119 reduced the proliferation of Th17 and phosphorylated STAT3 expression while increasing Treg differentiation and phosphorylated STAT5 expression. Moreover, STX0119 decreased the expression of IL-6 and -17 but not IL-10. These findings suggest that STX0119 can be used to treat autoimmune RA through inhibiting the activation of STAT3.
Animals ; Arthritis* ; Arthritis, Rheumatoid ; Autoimmune Diseases ; Cytokines ; In Vitro Techniques ; Inflammation ; Interleukin-10 ; Interleukin-17 ; Interleukin-6 ; Interleukins ; Joints ; Mice* ; STAT3 Transcription Factor ; Up-Regulation ; Zymosan

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Effect of Mesenchymal Stem Cells and Platelet-Derived Growth Factor on the Healing of Radiation Induced Ulcer in Rats.

Im Geon JIN ; Jin Ho KIM ; Hong Gyun WU ; Soon Jung HWANG

Tissue Engineering and Regenerative Medicine.2016;13(1):78-90. doi:10.1007/s13770-015-0055-x

Radiation-induced skin ulceration is a frequent complication of radiation therapy. This study investigated the effects of rat mesenchymal stem cells (rMSCs) and platelet-derived growth factor (PDGF) on the healing of radiation-induced soft tissue injury. Sprague-Dawley rats (n=17) were irradiated on the right and left buttocks with a single dose of 50 Gy. The right buttocks were administered with phosphate-buffered solution as a control. The left buttocks were administered with either rMSCs (2×10⁶ cells), PDGF (8 µg), or PDGF combined with rMSCs. Administration was done at three weeks after irradiation. Wound healing was analyzed by calculating the percentage of residual ulcerated skin area compared to the total irradiated area during the five week healing period after administration. Modified skin scores were also assessed. Finally, skin lesions were histologically evaluated. More than 40% of the irradiated skin area within the irradiated zone underwent ulceration within 16 days postirradiation, with peak ulceration exceeding 50% around three weeks post-irradiation. Administration of rMSCs or PDGF alone did not confer any significant healing effect. The combined rMSCs+PDGF treatment significantly reduced the wound size compared with the nontreated control up to two weeks postinjection. Regarding the histological examination, lesions administered with PDGF (either alone or mixed with rMSCs) resulted in a greater deposition of highly organized collagen fibers throughout the dermis layer, compared with the control. In conclusion, the combined administration of rMSCs and PDGF efficiently enhanced the healing of radiation-induced skin ulceration.
Animals ; Buttocks ; Collagen ; Dermis ; Mesenchymal Stromal Cells* ; Platelet-Derived Growth Factor* ; Rats* ; Rats, Sprague-Dawley ; Skin ; Skin Ulcer ; Soft Tissue Injuries ; Ulcer* ; Wound Healing ; Wounds and Injuries

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Histomorphometric Evaluation of Onlay Freeze-Dried Block Bone and Deproteinized Bovine Bone with Collagen in Rat.

Gyu Un JUNG ; Seong Jin HONG ; Ji Youn HONG ; Eun Kyoung PANG

Tissue Engineering and Regenerative Medicine.2016;13(1):70-77. doi:10.1007/s13770-016-9021-0

The aim of this study was to evaluate the effect of human freeze-dried bone block (FDBB) and deproteinized bovine bone with collagen (DBBC) on bone formation when applied as an onlay graft in rat calvariums. Thirty male Sprague-Dawley rats received collagen sponge (control), FDBB, or DBBC onlay grafts trimmed into 8-mm disks measuring 4-mm height. Each graft was secured onto the calvarium surface using horizontal mattress sutures. Rats in each group were killed at 2 (n=5) or 8 (n=5) weeks postoperatively for histologic and histomorphometric analysis. The total augmented area (mm²), new bone area (mm²), and bone density (%) were measured. The FDBB and DBBC groups showed significantly more new bone formation and bone density than the control group at 2 and 8 weeks. The increased new bone area was significantly greater in the FDBB group than in the DBBC group (p<0.05). The total augmented area was significantly higher in the FDBB and DBBC groups at 2 and 8 weeks than in the control group (p<0.05), and at 8 weeks, the area was significantly decreased in the DBBC group compared to that in the FDBB group and the area at 2 weeks (p<0.05). Within the limitations of the present study, we concluded that onlay FDBB and DBBC grafts caused new bone formation through an osteoconductive mechanism. In addition, compared to FDBB, DBBC had less capacity to form new bone and maintain the space.
Animals ; Bone Density ; Collagen* ; Humans ; Inlays* ; Male ; Osteogenesis ; Porifera ; Rats* ; Rats, Sprague-Dawley ; Skull ; Space Maintenance, Orthodontic ; Sutures ; Transplants

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Construction of a New T-Vector: Nickase (Nt.BspQI)-Generated T-Vector Bearing a Reddish-Orange Indicator Gene.

Ji Young CHOI ; Chulman JO ; Sangmee Ahn JO

Tissue Engineering and Regenerative Medicine.2016;13(1):66-69. doi:10.1007/s13770-015-0118-z

T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT^GAAGAGC-3′) at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3′-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.
Clone Cells ; Cloning, Organism ; Deoxyribonuclease I* ; Methods ; Myostatin ; Plasmids ; Polymerase Chain Reaction

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Extracellular Calcium-Binding Peptide-Modified Ceramics Stimulate Regeneration of Calvarial Bone Defects.

Ju Ang KIM ; Young Ae CHOI ; Hui Suk YUN ; Yong Chul BAE ; Hong In SHIN ; Eui Kyun PARK

Tissue Engineering and Regenerative Medicine.2016;13(1):57-65. doi:10.1007/s13770-015-9066-x

Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.
Amino Acids ; Animals ; Bone Marrow ; Bone Regeneration ; Calcium ; Ceramics* ; Collagen Type I ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice ; Miners ; Osteocalcin ; Osteogenesis ; Photoelectron Spectroscopy ; Regeneration* ; X-Ray Microtomography

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Rapid Expansion and Auto-Grafting Efficiency of Porcine Full Skin Expanded by a Skin Bioreactor Ex Vivo.

Man Il HUH ; Sun Hee AN ; Hwi Gang KIM ; Yun Jeong SONG ; Eun Chang CHOI ; Sang Hyun AN ; Woo Sung CHOI ; Jeung Soo HUH ; Jeong Ok LIM

Tissue Engineering and Regenerative Medicine.2016;13(1):31-38. doi:10.1007/s13770-016-9096-0

Full skin auto-grafts are required for reconstruction of skin burns and trauma scars. However, currently available clinical approaches such as sheet skin graft, mesh skin grafts, artificial skin graft, and in vivo skin expansion have limitations due to their potential danger for secondary damage and scar formation at the donor site, and discomfort during skin expansion. We developed an advanced bioreactor system and evaluated its function in skin expansion using porcine full skin. The reactor was designed as a pneumatic cylinder type, was programmed to adjust the pressure and the operating time. The system was composed of culture chamber unit, environmental control unit, and monitoring unit. Skins were expanded at 200 kPa pneumatic force and the expanded skins were analyzed by immunohistochemistry and histology. Furthermore we carried out auto-grafting experiment of the expanded skins in vivo using Yucatan pigs and skins were harvested and histologically analyzed after 8 weeks. The results showed that the bioreactor expanded skins to 160% in 4 hours. Histological analysis of the expanded skins revealed that epidermal cells and dermal fibroblasts were viable and remained integrity. The results of auto-grafting experiment indicated that fibrosis and scars were not detected in the grafted skins. This study demonstrates that the newly developed skin bioreactor enabled to obtain large sized full skin rapidly and successful grating.
Bioreactors* ; Burns ; Cicatrix ; Fibroblasts ; Fibrosis ; Humans ; Immunohistochemistry ; Skin* ; Skin, Artificial ; Swine ; Tissue Donors ; Transplants

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Effect of Fibroblast Growth Factor-2 and Retinoic Acid on Lineage Commitment of Bone Marrow Mesenchymal Stem Cells.

Jiwon LIM ; Eui Kyun PARK

Tissue Engineering and Regenerative Medicine.2016;13(1):47-56. doi:10.1007/s13770-016-9102-0

In this study, we examined the effect of a combination of fibroblast growth factor-2 (FGF-2) and retinoic acid (RA) on osteoblast and adipocyte lineage commitment and differentiation of human bone marrow mesenchymal stem cells (BMSCs). Pretreatment of human BMSCs with FGF-2 or RA for 5 days followed by osteoblast differentiation induction showed high calcium deposition compared to control. A combination of FGF-2 and RA further induced calcium deposition compared to FGF-2 or RA alone. The enhanced mineral deposition was accompanied with the increased expression of osteoblast differentiation markers, alkaline phosphatase and osteocalcin. On the other hand, FGF-2 pretreatment followed by adipocyte differentiation induction also showed increased formation of lipid droplets in human BMSCs, whereas RA pretreatment suppressed formation of lipid droplets. However, a combination of FGF-2 and RA increased formation of lipid droplets and expression of adipocyte marker genes, including adiponectin, ADIPOQ, FABP4, peroxisome proliferator-activated receptor γ (PPARγ), and C/EBPα. During pretreatment of BMSCs with FGF-2, RA or in combination, the cells expressed similar levels of MSC surface markers such as CD29, CD44, CD90, and CD105, indicating that they maintain stem cell potential. To determine how RA cooperates with FGF-2 in osteoblast and adipocyte lineage commitment, the expression of RA receptors and intracellular lipid-binding proteins was examined. A combination of FGF-2 and RA strongly induced the expression of RA receptor α, β, γ, PPAR β/δ, CRABP-II, and FABP5. Collectively, these results demonstrate that combined pretreatment of human BMSCs with FGF-2 and RA enhances the commitment into osteoblast and adipocyte lineages through modulation of the expression of RA-related genes.
Adipocytes ; Adiponectin ; Alkaline Phosphatase ; Antigens, Differentiation ; Bone Marrow* ; Calcium ; Fibroblast Growth Factor 2* ; Fibroblasts* ; Hand ; Humans ; Lipid Droplets ; Mesenchymal Stromal Cells* ; Miners ; Osteoblasts ; Osteocalcin ; Peroxisome Proliferator-Activated Receptors ; Peroxisomes ; Stem Cells ; Tretinoin*

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Characterization of Adipose Tissue Mesenchymal Stromal Cell Subsets with Distinct Plastic Adherence.

Jung Hyun PARK ; Ki Joo KIM ; Jong Won RHIE ; Il Hoan OH

Tissue Engineering and Regenerative Medicine.2016;13(1):39-46. doi:10.1007/s13770-015-0027-1

Mesenchymal stromal cells (MSCs) established by in-vitro adherence culture have been widely utilized for various cell therapeutic trials, but potential heterogeneity that can be caused by preparation methods are poorly characterized. In this study, we show that at least two distinct subsets of MSCs with different adherence to plastic surface exist in human adipose tissue-derived stromal vascular fraction (SVF); while 69% of total colony forming units in SVF adhere to the surface before 3 hrs of plating, 13–17% of colonogenic cells adhered to the surface at later period of 15 hr to 1 week after plating. Of note, the late adherent MSCs exhibited higher self-renewal of colony forming cells and higher proliferating potential with comparable level of osteogenic or adipogenic differentiation potential to the early adherence subsets. Moreover, late adherent cells exhibited distinct pattern of paracrine secretome including higher level secretion of cytokines than the early adherent subsets. Taken together, these results suggest the possibility that distinct adherence properties of MSCs can be another parameter of clonal heterogeneity in the subpopulations of adipose tissue MSCs and that it can be an important factor for optimization of MSC preparation for cell therapeutic trials.
Adipose Tissue* ; Cytokines ; Humans ; Mesenchymal Stromal Cells* ; Plastics* ; Population Characteristics ; Stem Cells

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In Vitro Performance of Injectable Chitosan-Tripolyphosphate Scaffolds Combined with Platelet-Rich Plasma.

Andréa Arruda Martins SHIMOJO ; Sofia Elisa Moraga GALDAMES ; Amanda Gomes Marcelino PEREZ ; Thiago Heiji ITO ; Angela Cristina MALHEIROS LUZO ; Maria Helena Andrade SANTANA

Tissue Engineering and Regenerative Medicine.2016;13(1):21-30. doi:10.1007/s13770-015-9111-9

This study aimed to evaluate the in vitro biological effectiveness of chitosan microparticles crosslinked with sodium tripolyphosphate (TPP) in combination with activated pure platelet-rich plasma (aP-PRP) as an injectable composite scaffold for growth factors release, cell proliferation and osteogenic differentiation. Two main novelties were addressed in the field of scaffolds in regenerative medicine: the first is the approach including simultaneously the three vertices of the proliferation triangle formed by the capabilities genic progenitor cells, conductive scaffolds and inductive growth factors, which are provided by platelet rich plasma (PRP); secondly, the approach of an injectable composite scaffolds formed by the fibrin network from aP-PRP and the chitosan microparticles crosslinked with TPP. The microparticles were prepared by vortexing the chitosan and TPP solutions. The ionic crosslinking of chitosan with TPP was made at mass ratios of 2:1, 5:1, and 10:1 at pH 4.0. P-PRP was obtained via the controlled centrifugation of whole blood. The composite scaffolds were prepared by adding the microparticles to immediately activated P-PRP. The results showed that the microparticles had adequate physicochemical and mechanical properties for injection. Furthermore, the microparticles controlled the release of growth factors from P-PRP. The proliferation of human adipose-derived mesenchymal stem cells was lower than in aP-PRP alone but significant at a 2:1 chitosan-TPP mass ratio. Osteogenic differentiation was stimulated at all studied mass ratios, as indicated by the alkaline phosphatase activity. These results offer perspectives for optimizing the composite scaffold, and to prove its potential as an injectable scaffold in regenerative medicine.
Alkaline Phosphatase ; Cell Proliferation ; Centrifugation ; Chitosan ; Fibrin ; Humans ; Hydrogen-Ion Concentration ; In Vitro Techniques* ; Intercellular Signaling Peptides and Proteins ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma* ; Regenerative Medicine ; Sodium ; Stem Cells

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