Byung-Jae Kang

Comparison of canine umbilical cord blood-derived mesenchymal stem cell transplantation times: involvement of astrogliosis, inflammation, intracellular actin cytoskeleton pathways, and neurotrophin-3.

Canine mesenchymal stem cells (cMSCs) derived from umbilical cord blood represent a potentially useful source of stem cells for therapy. The aim of this study was to compare the effects of different transplantation times of cMSCs after spinal cord injury (SCI). A total of 21 dogs were subjected to SCI by balloon-induced compression of the first lumbar vertebrae for 12 h. Of the 21 dogs, 12 were divided into four groups of three according to the time of stem cell (1 × 106) transplantation at the injury site: control no treatment, 12 h, 1 week, and 2 weeks. The remaining 9 animals were negative harvest (HA) time controls for each treatment group (n = 3). Olby and Tarlov scores were used to evaluate functional recovery of the hindlimbs. Markers for neuronal regeneration (Tuj-1, nestin, MAP2, and NF-M), astrogliosis (GALC, GFAP, and pSTAT3), signal molecules for actin cytoskeleton (RhoA, Cdc42, and Rac1), inflammation (COX-2), and neurotrophins (NT-3) were evaluated by Western blot analysis. Scores of the 1-week transplantation group showed significant improvement compared to controls. Hematoxylin and eosin (H&E) staining revealed less fibrosis at the injury site in the 1-week transplantation group compared to other groups and immunohistochemistry showed increased expression of neuronal markers. Furthermore, in both 1-week and 2-week transplantation groups, Tuj-1, nestin, MAP2, NF-M, NT-3, and GFAP increased, but pSTAT3, GALC, and COX2 decreased. RhoA decreased and Rac1 and Cdc42 increased in the 1-week transplantation group. In conclusion, transplantation of cMSCs 1 week after SCI was more effective in improving clinical signs and neuronal regeneration and reducing fibrosis formation compared to the other transplantation times evaluated. Subsequently, these data may contribute to the optimization of timing for MSC transplantation used as a therapeutic modality.

Umbilical-cord-blood-derived mesenchymal stem cells seeded onto fibronectin-immobilized polycaprolactone nanofiber improve cardiac function

Stem cells seeded onto biofunctional materials have greater potency for therapeutic applications. We investigated whether umbilical-cord-blood-derived mesenchymal stem cell (UCB-MSC)-seeded fibronectin (FN)-immobilized polycaprolactone (PCL) nanofibers could improve cardiac function and inhibit left ventricle (LV) remodeling in a rat model of myocardial infarction (MI). Aligned nanofibers were uniformly coated with poly(glycidyl methacrylate) by initiated chemical vapor deposition followed by covalent immobilization of FN proteins. The degree of cell elongation and adhesion efficacy were improved by FN immobilization. Furthermore, genes related to angiogenesis and mesenchymal differentiations were up-regulated in the FN-immobilized PCL nanofibers in comparison to control PCL nanofibers in vitro. 4 weeks after the transplantation in the rat MI model, the echocardiogram showed that the UCB-MSC-seeded FN-immobilized PCL nanofiber group increased LV ejection fraction and fraction shortening as compared to the non-treated control and acellular FN-immobilized PCL nanofiber groups. Histological analysis indicated that the implantation of UCB-MSCs with FN-immobilized PCL nanofibers induced a decrease in MI size and fibrosis, and an increase in scar thickness. This study indicates that FN-immobilized biofunctional PCL nanofibers could be an effective carrier for UCB-MSC transplantation for the treatment of MI.

Injectable multifunctional microgel encapsulating outgrowth endothelial cells and growth factors for enhanced neovascularization

Recent cell-based therapy approaches have employed both nanotechnologies and other biomedical technologies to enhance their therapeutic potential. A combined strategy using therapeutic stem/progenitor cells and angiogenic proteins is attractive for the treatment of vascular disease. In this study, we developed an injectable multifunctional micro-sized gel system (microgel), composed of arginine-glycine-aspartic acid (RGD)-conjugated alginate, for the delivery of both cells and growth factors in vivo. The microgels encapsulated with outgrowth endothelial cells (OECs) and growth factors (vascular endothelial growth factor, VEGF, and hepatocyte growth factor, HGF) were formed via electrospraying. Cells encapsulated within the microgel exhibited a time-dependent proliferation with enhanced cell viability, and the size-controlled microgels resulted in sustained release of growth factors for enhanced new vessel formation by tube formation and rat aorta sprouting in vitro. Increased angiogenesis was also estimated in mice treated with RGD-microgel containing OECs and growth factors. Furthermore, injection of the multifunctional microgel into a hindlimb ischemia model improved blood flow perfusion and increased the capillary density by histological analysis. Compared with hydrogel system, injectable microgel system was shown to be superior with no toxicity. Overall, our injectable multifunctional microgel system can be attributed to deliver potential therapeutic agents/cells for the treatment of vascular diseases.

Low-level laser therapy promotes the osteogenic potential of adipose-derived mesenchymal stem cells seeded on an acellular dermal matrix.

This study investigates the feasibility of using an adipose-derived mesenchymal stem cell (ASC)-seeded acellular dermal matrix (ADM) along with low-level laser therapy (LLLT) to repair bone defect in athymic nude mice. Critical-sized calvarial defects were treated either with ADM, ADM/LLLT, ADM/ASCs, or ADM/ASCs/LLLT. In micro-computed tomography scans, the ADM/ASCs and the ADM/ASCs/LLLT groups showed remarkable bone formation after 14 days. Additionally, bone regeneration in the ADM/ASCs/LLLT group was obvious at 28 days, but in the ADM/ASCs group at 56 days. Bone mineral density and bone tissue volume in the ADM/ASCs/LLLT group significantly increased after 7 days, but in the ADM/ASCs group after 14 days. Histological analysis revealed that the defects were repaired in the ADM/ASCs and the ADM/ASCs/LLLT group, while the defects in the ADM and the ADM/LLLT groups exhibited few bone islands at 28 and 56 days. The successful seeding of ASCs onto ADM was confirmed, and LLLT enhanced the proliferation and the survival of ASCs at 14 days. Our results indicate that ASC-seeded grafts promote bone regeneration, and the application of LLLT on ASC-seeded ADM results in rapid bone formation. The implantation of an ASC-seeded ADM combined with LLLT may be used effectively for bone regeneration.

Targeted Genome Engineering to Control VEGF Expression in Human Umbilical Cord Blood‐Derived Mesenchymal Stem Cells: Potential Implications for the Treatment of Myocardial Infarction

Human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell‐based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF‐secreting stem cell (VEGF/hUCB‐MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB‐MSC chromosome, the transcription activator‐like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB‐MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB‐MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB‐MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB‐MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB‐MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF‐secreting stem cell system is an effective therapeutic approach for the treatment of MI. Stem Cells Translational Medicine 2017;6:1040–1051

Comparison of Osteogenesis between Adipose-Derived Mesenchymal Stem Cells and Their Sheets on Poly-ε-Caprolactone/β-Tricalcium Phosphate Composite Scaffolds in Canine Bone Defects

Multipotent mesenchymal stem cells (MSCs) and MSC sheets have effective potentials of bone regeneration. Composite polymer/ceramic scaffolds such as poly-ε-caprolactone (PCL)/β-tricalcium phosphate (β-TCP) are widely used to repair large bone defects. The present study investigated the in vitro osteogenic potential of canine adipose-derived MSCs (Ad-MSCs) and Ad-MSC sheets. Composite PCL/β-TCP scaffolds seeded with Ad-MSCs or wrapped with osteogenic Ad-MSC sheets (OCS) were also fabricated and their osteogenic potential was assessed following transplantation into critical-sized bone defects in dogs. The alkaline phosphatase (ALP) activity of osteogenic Ad-MSCs (O-MSCs) and OCS was significantly higher than that of undifferentiated Ad-MSCs (U-MSCs). The ALP, runt-related transcription factor 2, osteopontin, and bone morphogenetic protein 7 mRNA levels were upregulated in O-MSCs and OCS as compared to U-MSCs. In a segmental bone defect, the amount of newly formed bone was greater in PCL/β-TCP/OCS and PCL/β-TCP/O-MSCs/OCS than in the other groups. The OCS exhibit strong osteogenic capacity, and OCS combined with a PCL/β-TCP composite scaffold stimulated new bone formation in a critical-sized bone defect. These results suggest that the PCL/β-TCP/OCS composite has potential clinical applications in bone regeneration and can be used as an alternative treatment modality in bone tissue engineering.

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28 days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p < 0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.

Evaluation of Mesenchymal Stem Cell Sheets Overexpressing BMP-7 in Canine Critical-Sized Bone Defects

The aim of this study was to investigate the in vitro osteogenic capacity of bone morphogenetic protein 7 (BMP-7) overexpressing adipose-derived (Ad-) mesenchymal stem cells (MSCs) sheets (BMP-7-CS). In addition, BMP-7-CS were transplanted into critical-sized bone defects and osteogenesis was assessed. BMP-7 gene expressing lentivirus particles were transduced into Ad-MSCs. BMP-7, at the mRNA and protein level, was up-regulated in BMP-7-MSCs compared to expression in Ad-MSCs. Osteogenic and vascular-related gene expressions were up-regulated in BMP-7-CS compared to Ad-MSCs and Ad-MSC sheets. In a segmental bone-defect model, newly formed bone and neovascularization were enhanced with BMP-7-CS, or with a combination of BMP-7-CS and demineralized bone matrix (DBM), compared to those in control groups. These results demonstrate that lentiviral-mediated gene transfer of BMP-7 into Ad-MSCs allows for stable BMP-7 production. BMP-7-CS displayed higher osteogenic capacity than Ad-MSCs and Ad-MSC sheets. In addition, BMP-7-CS combined with demineralized bone matrix (DBM) stimulated new bone and blood vessel formation in a canine critical-sized bone defect. The BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. Thus, BMP-7-CS and DBM have therapeutic potential for the treatment of critical-sized bone defects and could be used to further enhance clinical outcomes during bone-defect treatment.