Japanese Laws and the Current Status of Regenerative Medicine in the Tohoku Region.

AIM: The aim of this study was to review Japanese laws regarding regenerative medicine and the current status of clinical application of regenerative medicine, to learn about the advantages and problems, and to thereby serve as a reference for measures necessary for the development of regenerative medicine. BACKGROUND: Regenerative medicine started in 1957 with the transplantation of hematopoietic stem cells, followed by the establishment of embryonic stem cells in 1981 and induced pluripotent stem cells in 2006, and continues to evolve progressively. At the same time, however, problems have emerged due to lax legal regulations, such as the use of treatments that lack scientific evidence. REVIEW RESULTS: The Japanese government enacted two laws to regulate regenerative medicine: the Law to Ensure the Safety of Regenerative Medicine and the Amend the Pharmaceutical Affairs Law in 2013. These laws were enacted with the aim of providing safe regenerative medicine promptly and smoothly and developing many regenerative medicine products. In these laws, regenerative medicine is defined as medical treatment that restores lost functions of damaged organs and tissues with the help of cellular and tissue-based products. Nowadays, there are two major methods of regenerative medicine. One representative method involves the transplantation of devices that activates self-regenerative ability by introducing living cells into patients' body. The other method is the activation and differentiation of endogenous stem cells with cell growth and differentiation factors. CONCLUSION: The current status of regenerative medicine in the Tohoku region after the enactment of these laws is described in detail. This clarified the advantages and disadvantages associated with regenerative medicine as it is currently practiced in Japan. CLINICAL SIGNIFICANCE: Development of regenerative medicine in dentistry will be advanced by learning about its clinical application in medicine.

Rapid and efficient generation of cartilage pellets from mouse induced pluripotent stem cells by transcriptional activation of BMP-4 with shaking culture.

Induced pluripotent stem cells (iPSCs) offer an unlimited source for cartilage regeneration as they can generate a wide spectrum of cell types. Here, we established a tetracycline (tet) controlled bone morphogenetic protein-4 (BMP-4) expressing iPSC (iPSC-Tet/BMP-4) line in which transcriptional activation of BMP-4 was associated with enhanced chondrogenesis. Moreover, we developed an efficient and simple approach for directly guiding iPSC-Tet/BMP-4 differentiation into chondrocytes in scaffold-free cartilaginous pellets using a combination of transcriptional activation of BMP-4 and a 3D shaking suspension culture system. In chondrogenic induction medium, shaking culture alone significantly upregulated the chondrogenic markers Sox9, Col2a1, and Aggrecan in iPSCs-Tet/BMP-4 by day 21. Of note, transcriptional activation of BMP-4 by addition of tet (doxycycline) greatly enhanced the expression of these genes. The cartilaginous pellets derived from iPSCs-Tet/BMP-4 showed an oval morphology and white smooth appearance by day 21. After day 21, the cells presented a typical round morphology and the extracellular matrix was stained intensively with Safranin O, alcian blue, and type II collagen. In addition, the homogenous cartilaginous pellets derived from iPSCs-Tet/BMP-4 with 28 days of induction repaired joint osteochondral defects in immunosuppressed rats and integrated well with the adjacent host cartilage. The regenerated cartilage expressed the neomycin resistance gene, indicating that the newly formed cartilage was generated by the transplanted iPSCs-Tet/BMP-4. Thus, our culture system could be a useful tool for further investigation of the mechanism of BMP-4 in regulating iPSC differentiation toward the chondrogenic lineage, and should facilitate research in cartilage development, repair, and osteoarthritis.

Titanium surface with nanospikes tunes macrophage polarization to produce inhibitory factors for osteoclastogenesis through nanotopographic cues.

Definitive prevention of inflammatory osteolysis around peri-implant bone tissue remains unestablished. M1 macrophages play a key role in the host defense against inflammatory osteolysis, and their polarization depends on cell shape. Macrophage polarization is controlled by environmental stimuli, particularly physicochemical cues and hence titanium nanosurface might tune macrophage polarization and function. This study determined whether titanium nanosurfaces with anisotropically patterned nanospikes regulates macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors. Alkaline-etching treatment with different protocols created two types of titanium nanosurfaces that had anisotropically patterned nanospikes with high or low distribution density, together with superhydrophilicity and the presence of hydroxyl groups. J774A.1 cells (mouse macrophage-like cell line), cultured on both titanium nanosurfaces, exhibited truly circulated shapes and highly expressed M1, but less M2, markers, without loss of viability. M1-like polarization of macrophages on both titanium nanosurfaces was independent of protein-mediated ligand stimulation or titanium surface hydrophilic or chemical status. In contrast, other smooth or micro-roughened titanium surfaces with little or no nanospikes did not activate macrophages under any culture conditions. Macrophage culture supernatants on both titanium nanosurfaces inhibited osteoclast differentiation of RAW264.7 cells (mouse osteoclast precursor cell line), even when co-incubated with osteoclast differentiation factors. The inhibitory effects on osteoclast differentiation tended to be higher in macrophages cultured on titanium nanosurfaces with denser nanospikes. These results showed that titanium nanosurfaces with anisotropically patterned nanospikes tune macrophage polarization for inhibiting osteoclast differentiation of osteoclast precursors, with nanotopographic cues rather than other physicochemical properties. STATEMENT OF SIGNIFICANCE: Peri-implant inflammatory osteolysis is one of the serious issues for dental and orthopedic implants. Macrophage polarization and function are key for prevention of peri-implant inflammatory osteolysis. Macrophage polarization can be regulated by the biomaterial's surface physicochemical properties such as hydrophilicity or topography. However, there was no titanium surface modification to prevent inflammatory osteolysis through immunomodulation. The present study showed for the first time that the titanium nanosurfaces with anisotropically patterned nanospikes, created by the simple alkali-etching treatment polarized macrophages into M1-like type producing the inhibitory factor on osteoclast differentiation. This phenomenon attributed to nanotopographic cues, but not hydrophilicity on the titanium nanosurfaces. This nanotechnology might pave the way to develop the smart implant surface preventing peri-implant inflammatory osteolysis through immunomodulation.

Gene transfection achieved by utilizing antibacterial calcium phosphate nanoparticles for enhanced regenerative therapy.

Protamine-coated multi-shell calcium phosphate (CaP) was developed as a non-viral vector for tissue regeneration therapy. CaP nanoparticles loaded with different amounts of plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) were used to treat MC3T3E1 cells, and the yield of the released BMP-2 or IGF-1 was measured using ELISA 3 days later. Collagen scaffolds containing CaP nanoparticles were implanted into rat cranial bone defects, and BMP-2 and IGF-1 yields, bone formation, and bone mineral density enhancement were evaluated 28 days after gene transfer. The antibacterial effects of CaP nanoparticles against Streptococcus mutans and Aggregatibacter actinomycetemcomitans increased with an increase in the protamine dose, while they were lower for Staphylococcus aureus and Porphyromonas gingivalis. In the combination treatment with BMP-2 and IGF-1, the concentration ratio of BMP-2 and IGF-1 is an important factor affecting bone formation activity. The calcification activity and OCN mRNA of MC3T3E1 cells subjected to a BMP-2:IGF-1 concentration ratio of 1:4 was higher at 14 days. During gene transfection treatment, BMP-2 and IGF-1 were released simultaneously after gene transfer; the loaded dose of the plasmid DNA encoding IGF-1 did not impact the BMP-2 or IGF-1 yield or new bone formation ratio in vitro and in vivo. In conclusion, two growth factor-releasing systems were developed using an antibacterial gene transfer vector, and the relationship between the loaded plasmid DNA dose and resultant growth factor yield was determined in vitro and in vivo.

Prolonged release of bone morphogenetic protein-2 in vivo by gene transfection with DNA-functionalized calcium phosphate nanoparticle-loaded collagen scaffolds.

In the combination of scaffolds immersed in growth factor solutions, the release of growth factors mainly depends on scaffold degradation. However, the release of bone morphogenetic protein (BMP)-2 at an appropriate concentration during the stage of tissue regeneration would enhance bone regeneration. To achieve this condition, the present study was performed to investigate the effects of scaffolds combined with gene transfection using non-viral vectors. Nanohydroxyapatite-collagen (nHAC) scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or ascorbic acid/copper chloride, and a collagen scaffold (Terdermis®) were prepared, loaded with BMP-2-encoding plasmid DNA-functionalized calcium phosphate nanoparticles (CaP), naked plasmid DNA, or BMP-2 solution, and implanted in rats. The yield of released BMP-2 and its releasing period, respectively, were larger and longer from the scaffolds loaded with CaP than from those incubated with BMP-2 solution. In addition, the alkaline phosphatase activity induced by the CaP-loaded scaffolds was higher. Histological analysis showed that released BMP-2 could be observed on the macrophages or multinuclear giant cells surrounding the nHAC fragments or collagen fibres. TRAP-positive or OCN-positive sites were observed in all groups and a mineralization area was observed in the Terdermis®/CaP sample. The present study demonstrates that gene transfection by scaffold loaded with CaP gene transfer vectors induces a larger yield of BMP-2 for a longer period than by scaffolds loaded with BMP-2 solution or naked plasmid.

Binding of PICK1 PDZ domain with calcineurin B regulates osteoclast differentiation.

The calcineurin/nuclear factor of activated T cell (NFAT) signaling pathway plays a major role in osteoclast differentiation; however, the proteins that react with the calcineurin-NFAT complex in osteoclasts to regulate osteoclastogenesis remain unclear. Here, we present evidence that PICK1 also positively regulates calcineurin B in osteoclasts to activate NFAT to promote osteoclastogenesis. mRNA and protein expression of PICK1 in murine primary bone marrow macrophages (BMMs) was significantly increased during RANKL-induced osteoclast differentiation. The interaction of PICK1 with calcineurin B in BMMs was confirmed by co-immunoprecipitation. An inhibitor of the PICK1 PDZ domain significantly decreased osteoclastogenesis marker gene expression and the number of TRAP-positive multinucleated cells among RAW264.7 osteoclast progenitor cells. Overexpression of PICK1 in RAW264.7 cells significantly increased the number of TRAP-positive mature osteoclasts. Increased NFAT activation with transcriptional activation of PICK1 during RAW264.7 osteoclastogenesis was also confirmed in a tetracycline-controlled PICK1 expression system. These results suggest that the PDZ domain of PICK1 directly interacts with calcineurin B in osteoclast progenitor cells and promotes osteoclast differentiation through activation of calcineurin-NFAT signaling.

Amiloride-enhanced gene transfection of octa-arginine functionalized calcium phosphate nanoparticles.

Nanoparticles represent promising gene delivery systems in biomedicine to facilitate prolonged gene expression with low toxicity compared to viral vectors. Specifically, nanoparticles of calcium phosphate (nCaP), the main inorganic component of human bone, exhibit high biocompatibility and good biodegradability and have been reported to have high affinity for protein or DNA, having thus been used as gene transfer vectors. On the other hand, Octa-arginine (R8), which has a high permeability to cell membrane, has been reported to improve intracellular delivery systems. Here, we present an optimized method for nCaP-mediated gene delivery using an octa-arginine (R8)-functionalized nCaP vector containing a marker or functional gene construct. nCaP particle size was between 220-580 nm in diameter and all R8-functionalized nCaPs carried a positive charge. R8 concentration significantly improved nCaP transfection efficiency with high cell compatibility in human mesenchymal stem cells (hMSC) and human osteoblasts (hOB) in particular, suggesting nCaPs as a good option for non-viral vector gene delivery. Furthermore, pre-treatment with different endocytosis inhibitors identified that the endocytic pathway differed among cell lines and functionalized nanoparticles, with amiloride increasing transfection efficiency of R8-functionalized nCaPs in hMSC and hOB.

Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells.

Three-dimensional (3D) cell constructs are expected to provide osteoinductive materials to develop cell-based therapies for bone regeneration. The proliferation and spontaneous aggregation capability of induced pluripotent stem cells (iPSCs) thus prompted us to fabricate a scaffold-free iPSC construct as a transplantation vehicle. Embryoid bodies of mouse gingival fibroblast-derived iPSCs (GF-iPSCs) were seeded in a cell chamber with a round-bottom well made of a thermoresponsive hydrogel. Collected ball-like cell constructs were cultured in osteogenic induction medium for 30 days with gentle shaking, resulting in significant upregulation of osteogenic marker genes. The constructs consisted of an inner region of unstructured cell mass and an outer osseous tissue region that was surrounded by osteoblast progenitor-like cells. The outer osseous tissue was robustly calcified with elemental calcium and phosphorous as well as hydroxyapatite. Subcutaneous transplantation of the GF-iPSC constructs into immunodeficient mice contributed to extensive ectopic bone formation surrounded by teratoma tissue. These results suggest that mouse GF-iPSCs could facilitate the fabrication of osteoinductive scaffold-free 3D cell constructs, in which the calcified regions and surrounding osteoblasts may function as scaffolds and drivers of osteoinduction, respectively. With incorporation of technologies to inhibit teratoma formation, this system could provide a promising strategy for bone regenerative therapies.

Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation of Amelx was associated with enhanced osteogenic differentiation, especially in the early stage of biomineralization.

PIH1D1 interacts with mTOR complex 1 and enhances ribosome RNA transcription.

PIH1D1 is the defining component of the R2TP complex. Recently, R2TP has been reported to stabilize mTOR (mammalian target of rapamycin), an important regulator of cell growth and protein synthesis. Two complexes of mTOR, mTORC1 and mTORC2, have been identified. We demonstrate that immunoprecipitation (IP) of PIH1D1 results in the co-IP of Raptor (mTORC1 specific), but not Rictor (mTORC2 specific), and that knockdown of PIH1D1 decreases mTORC1 assembly, S6 kinase phosphorylation (indicator of mTORC1 activity), and rRNA transcription without affecting mTORC2 in human breast cancer MCF-7 cells. In addition, we provide evidence that PIH1D1 is overexpressed in various breast cancer cell lines. These findings collectively suggest that PIH1D1 may have an important role in mTORC1 regulation in breast cancers.

Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

RPAP3 splicing variant isoform 1 interacts with PIH1D1 to compose R2TP complex for cell survival.

We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex.