The mechanism of thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides.

In this study, we investigated thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides (Chol-ASO). First, we evaluated platelet activation induced by Chol-ASO in mice by flow cytometry after administration of platelet-rich plasma (PRP). An increase in the number of large particle-size events with platelet activation was detected in the Chol-ASO-treated group. In a smear study, numerous platelets were observed to attach to nucleic acid-containing aggregates. A competition binding assay showed that the conjugation of cholesterol to ASOs increased their affinity for glycoprotein VI. Platelet-free plasma was then mixed with Chol-ASO to form aggregates. The assembly of Chol-ASO was confirmed by dynamic light scattering measurements in the concentration range in which the formation of aggregates with plasma components was observed. In conclusion, the mechanism by which Chol-ASOs causes thrombocytopenia is proposed to be as follows: (1) Chol-ASOs form polymers, (2) the nucleic acid portion of the polymers interacts with plasma proteins and platelets, which cross-links them to form aggregates, and (3) platelets bound to aggregates become activated, resulting in platelet aggregation, leading to a decrease in platelet count in vivo. The details of the mechanism revealed in this study could contribute to creating safer oligonucleotide therapies without the risk of thrombocytopenia.

Discovery of Brain-Penetrant Glucosylceramide Synthase Inhibitors with a Novel Pharmacophore.

Inhibition of glucosylceramide synthase (GCS) is a major therapeutic strategy for Gaucher's disease and has been suggested as a potential target for treating Parkinson's disease. Herein, we report the discovery of novel brain-penetrant GCS inhibitors. Assessment of the structure-activity relationship revealed a unique pharmacophore in this series. The lipophilic ortho-substituent of aromatic ring A and the appropriate directionality of aromatic ring B were key for potency. Optimization of the absorption, distribution, metabolism, elimination, toxicity (ADMETox) profile resulted in the discovery of T-036, a potent GCS inhibitor in vivo. Pharmacophore-based scaffold hopping was performed to mitigate safety concerns associated with T-036. The ring opening of T-036 resulted in another potent GCS inhibitor with a lower toxicological risk, T-690, which reduced glucosylceramide in a dose-dependent manner in the plasma and cortex of mice. Finally, we discuss the structural aspects of the compounds that impart a unique inhibition mode and lower the cardiovascular risk.

Constructing vascularized hepatic tissue by cell-assembled viscous tissue sedimentation method and its application for vascular toxicity assessment.

In vitro Construction of the liver sinusoidal structure using artificial tissue is an important but worthwhile challenge, particularly for assessing the risk of diseases such as sinusoidal obstruction syndrome (SOS). Current models are unsuitable for evaluating the toxicity because of lacking sinusoidal capillary. In this study, we developed a vascularized hepatic tissue (VHT) using a unique tissue engineering technique, the cell assembled viscous tissue by sedimentation (CAViTs) method. The "viscous bodies" created using the CAViTs method exhibited significant self-assembly within 6 h after seeding, promoting cell-cell interaction. The level of albumin secreted by the VHT was four times higher than that of 2D-coculture and maintained for 1 month. The gene expression pattern of the VHT was closer to that of total human liver, compared with the 2D system. Quantitative evaluations of the vascular structure of VHT treated with two typical SOS-inducing compounds, monocrotaline and retrorsine, revealed higher sensitivity (IC50 = 40.35 µM), 19.92 times higher than the cell-viability assay. Thus, VHT represents an innovative in vitro model that mimics the vessel network structure and could become a useful tool for the early screening of compounds associated with a risk of vascular toxicity. STATEMENT OF SIGNIFICANCE: Mimicking sinusoidal structures in in vitro liver model is important to consider from the perspective of predicting hepatotoxicity such like sinusoidal obstruction syndrome (SOS). However, it was difficult to reconstruct the vascular structure within the hepatocyte-rich environment. In this study, we constructed a vascularized hepatic tissue in a high-throughput manner by a unique method using collagen and heparin, and evaluated its applicability to toxicity assessment. Vessel morphology analysis of the model treated by monocrotaline, which is a well-known SOS-inducing compound, could predict the toxicity with higher sensitivity. To the best of our knowledge, this is the first report to provide vascularized hepatic tissues using sinusoidal endothelial cells at least for demonstrating applicability to the evaluation of SOS induction risk.

Cell-based high-throughput screening for the evaluation of reactive metabolite formation potential.

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 µM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 µM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.

Video-based assessment of drug-induced effects on contractile motion properties using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Drug-induced inotropic change is a risk factor in drug development; thus, de-risking is desired in the early stages of drug development. Unlike proarrhythmic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), few in vitro models were validated to predict cardiac contractility. Motion field imaging (MFI), a high-resolution block matching-based optical flow technique, was expected to possess high quantitative predictivity in the detection of contraction speed. We aimed to establish an in vitro model to assess drug-induced contractile changes using hiPSC-CMs and MFI. METHODS: MFI was designed to noninvasively characterize cardiomyocyte contractile behavior by analyzing light microscope video images, and maximum contraction speed (MCS) was used as the index of contractility. Using MFI, 9 inactive compounds, 10 negative inotropes, and 10 positive inotropes were tested. Two negative chronotropes, ZD7288 and ivabradine, were also tested. To determine the sensitivity and specificity of the assay, the minimum effective concentration of the MCS was compared with the human effective total therapeutic concentration for 28 compounds in clinical use. RESULTS: For 8 negative and 8 positive inotropes, the effects observed in in vivo and clinical studies were detected in MFI assay. MFI assay showed negative chronotropic changes without inotropic changes. MFI assay presented sufficient specificity (83%) and sensitivity (88%), and RNA-sequence analysis provided an insight into the relationship between occurrence of the false compounds and target gene expression. DISCUSSION: We demonstrated the utility of MFI assay using hiPSC-CMs to assess drug-induced contractile function. These results will facilitate the de-risking of compounds during early drug development.

A pilot study to establish human T-cell leukemia virus type 1 (HTLV-1) carrier model using common marmoset (Callithrix jacchus).

BACKGROUND: For the diagnosis and treatment of adult T-cell leukemia/lymphoma (ATLL) caused by human T-lymphotropic virus type 1 (HTLV-1) are required therapeutic modalities urgently. Non-human primate models for ATLL would provide a valuable information for clinical studies. We did a pilot study to establish an ATLL non-human primate model using common marmosets (Callithrix jacchus). METHODS: We inoculated HTLV-1-producing MT-2 cells into 9-month-old marmosets, either intraperitoneally or intravenously. We next administrated MT-2 cells into 13-month-old marmosets under cyclosporine A (CsA) treatment to promote infection. HTLV-1 infection was determined by measuring HTLV-1 antibody titer in the common marmosets. RESULTS: The HTLV-1 antibody titer increased in the intraperitoneally inoculated marmoset with or without CsA treatment, and it kept over five 5 years though proviral copy number (proviral load, PVL) remained low throughout the study. CONCLUSION: We obtained HTLV-1 asymptomatic carriers of common marmosets by inoculating MT-2 cells.

Non-transmissible MV Vector with Segmented RNA Genome Establishes Different Types of iPSCs from Hematopoietic Cells.

Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.

High-Throughput Screening to Evaluate Inhibition of Bile Acid Transporters Using Human Hepatocytes Isolated From Chimeric Mice.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.

KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.

Pathway-Based Drug Repositioning for Cancers: Computational Prediction and Experimental Validation.

Developing drugs with anticancer activity and low toxic side-effects at low costs is a challenging issue for cancer chemotherapy. In this work, we propose to use molecular pathways as the therapeutic targets and develop a novel computational approach for drug repositioning for cancer treatment. We analyzed chemically induced gene expression data of 1112 drugs on 66 human cell lines and searched for drugs that inactivate pathways involved in the growth of cancer cells (cell cycle) and activate pathways that contribute to the death of cancer cells (e.g., apoptosis and p53 signaling). Finally, we performed a large-scale prediction of potential anticancer effects for all the drugs and experimentally validated the prediction results via three in vitro cellular assays that evaluate cell viability, cytotoxicity, and apoptosis induction. Using this strategy, we successfully identified several potential anticancer drugs. The proposed pathway-based method has great potential to improve drug repositioning research for cancer treatment.

Oncolytic coxsackievirus therapy as an immunostimulator.

Recently, the active development of oncolytic virotherapy has gathered attention. Enterovirus research seeks to better understand its pathogenicity. In particular, coxsackievirus A21 (CVA21) is a promising candidate for oncolytic virotherapy, and thus is the focus of many clinical trials. We have reported that coxsackievirus B3 (CVB3) had potent oncolytic activity for cancer, and induced immunogenic cell death of CVB3-infected cells. We then genetically engineered wild type CVB3 and successfully produced a novel recombinant CVB3-miRT, improving its safety by the introducing an organ-specific miRNA target sequence. We also developed the production method of CVB3 agent, and are conducting a clinical trial of CVB3 therapy for cancer patients. In this report, we review recent clinical progress in oncolytic virotherapy of CVA21 and clinical development of our CVB3.

Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.

Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell state were transfected using nonliposomal transfection reagents with plasmid DNA or mRNA encoding enhanced green fluorescent protein. We found that most cells (DNA > 90%; mRNA > 99%) were transfected without the loss of undifferentiated PSC marker expression or pluripotency. Moreover, we demonstrated an efficient gene editing method using transcription activator-like effector nucleases (TALENs) targeting the adenomatous polyposis coli (APC) gene. Our new method may improve hPSC gene transfer techniques, thus facilitating their use for human regenerative medicine.

Improved hematopoietic differentiation of primate embryonic stem cells by inhibition of the PI3K-AKT pathway under defined conditions.

Hematopoietic stem/progenitor cells (HSPCs) derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have potential therapeutic applications in humans. To assess the safety and efficacy of ESC/iPSC-based therapies, reliable animal models are required prior to their clinical application. The common marmoset (CM) was recently found to be a useful nonhuman primate animal model for drug development and safety assessment. However, a method for the efficient hematopoietic differentiation of CM ESCs has not been established. In this study, we developed a novel and efficient method for differentiating CM ESCs into hematopoietic cells by transiently inhibiting the phosphoinositide 3-kinase (PI3K)-Protein kinase B (AKT) pathway, a critical pathway that maintains the undifferentiated state of CM ESCs during embryoid body (EB) formation. Compared with controls, transient inhibition of the P13K-AKT pathway resulted in a threefold increase in the proportion of enriched CD34⁺ cells (p < 0.001) and an increase in the number of hematopoietic colonies on day 8 of CM EB cultures. Moreover, number of blast colonies, number of hematopoietic progenitor cell populations of CD34⁺CD117⁺, CD34⁺CD45⁺, and CD43⁺CD45⁺ cells, and expression of hematopoietic genes were increased by transient inhibition of the PI3K-AKT pathway. We also demonstrated that the hematopoietic progenitor cell population was increased by inhibition of PI3K in a human system. Our novel and efficient ESC differentiation method might be useful for preclinical research on human hematopoietic disorders and may be efficiently translated to human ESC/iPSC-based regenerative medicine.

Peripheral nerve-derived CXCL12 and VEGF-A regulate the patterning of arterial vessel branching in developing limb skin.

In developing limb skin, peripheral nerves provide a spatial template that controls the branching pattern and differentiation of arteries. Our previous studies indicate that nerve-derived VEGF-A is required for arterial differentiation but not for nerve-vessel alignment. In this study, we demonstrate that nerve-vessel alignment depends on the activity of Cxcl12-Cxcr4 chemokine signaling. Genetic inactivation of Cxcl12-Cxcr4 signaling perturbs nerve-vessel alignment and abolishes arteriogenesis. Further in vitro assays allow us to uncouple nerve-vessel alignment and arteriogenesis, revealing that nerve-derived Cxcl12 stimulates endothelial cell migration, whereas nerve-derived VEGF-A is responsible for arterial differentiation. These findings suggest a coordinated sequential action in which nerve Cxcl12 functions over a distance to recruit vessels to align with nerves, and subsequent arterial differentiation presumably requires a local action of nerve VEGF-A in the nerve-associated vessels.

The use of fluorescent indoline dyes for side population analysis.

Dye efflux assay evaluated by flow cytometry is useful for stem cell studies. The side population (SP) cells, characterized by the capacity to efflux Hoechst 33342 dye, have been shown to be enriched for hematopoietic stem cells (HSCs) in bone marrow. In addition, SP cells are isolated from various tissues and cell lines, and are also potential candidates for cancer stem cells. However, ultra violet (UV) light, which is not common for every flow cytometer, is required to excite Hoechst 33342. Here we showed that a fluorescent indoline dye ZMB793 can be excited by 488-nm laser, equipped in almost all the modern flow cytometers, and ZMB793-excluding cells showed SP phenotype. HSCs were exclusively enriched in the ZMB793-excluding cells, while ZMB793 was localized in cytosol of bone marrow lineage cells. The efflux of ZMB793 dye was mediated by ATP binding cassette (ABC) transporter Abcg2. Moreover, staining properties were affected by the side-chain structure of the dyes. These data indicate that the fluorescent dye ZMB793 could be used for the SP cell analysis.

Spi-B is critical for plasmacytoid dendritic cell function and development.

Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-B-deficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this uneven distribution was likely because of defective retainment of mature nondividing pDCs in the BM. The expression pattern of cell-surface molecules in Spi-B-deficient mice indicated the involvement of Spi-B in pDC development. The developmental defects of pDCs in Spi-B-deficient mice were more prominent in the BM than in the peripheral lymphoid organs and were intrinsic to pDCs. We conclude that Spi-B plays critical roles in pDC function and development.

Inflammation imaging by silica nanoparticles with antibodies orientedly immobilized.

The objective of this study is to design fluorescent nanoprobes for inflammation imaging. An antibody against CD11b expressed on the surfaces of mouse macrophages (anti-CD11b), was fluorescently labeled. Protein G, which has an ability to bind the Fc region of antibody, was conjugated onto the surface of silica nanoparticles (SiNP). Then, the fluorescent-labeled anti-CD11b was orientedly immobilized to the SiNP surface through the specific protein G-antibody interaction. After the intravenous injection of anti-CD11b orientedly immobilized SiNP to the mouse model of acute interstitial nephritis, unilateral ureteral obstruction (UUO) of one kidney, the fluorescent intensity at the UUO and non-treated, normal kidneys was assessed. The anti-CD11b orientedly immobilized SiNP were accumulated in the UUO kidney to a significantly great extent compared with the normal, non-inflamed kidney. The fluorescence intensity of inflamed kidney 6 and 12 h after injection of the anti-CD11b orientedly immobilized SiNP were significantly higher than that of anti-CD11b randomly immobilized SiNP or free anti-CD11b injection. Histological experiments revealed that the anti-CD11b orientedly immobilized SiNP were associated with macrophages infiltrated into the inflammation site. It is concluded that the anti-CD11b orientedly immobilized SiNP are promising nanoprobes to image the inflammation site at a high intensity.

Orientation-regulated immobilization of Jagged1 on glass substrates for ex vivo proliferation of a bone marrow cell population containing hematopoietic stem cells.

Notch signaling has been recognized as a key pathway to regulate the proliferation and differentiation of hematopoietic stem cells (HSC). In this study, the orientation-regulated immobilization of a Notch ligand was designed to achieve the efficient Notch ligand-receptor recognition for the ex vivo proliferation of a bone marrow cell population containing HSC. Protein A was chemically conjugated onto aminated glass substrates, followed by immobilizing a recombinant chimeric protein of Jagged1 and Fc domain (Jagged1-Fc) through the biospecific binding between protein A and Fc domain. Protein A adsorption was suppressed for the Jagged1-Fc-immobilized substrates, in contrast to the Jagged1-Fc-coated ones, indicating the orientation-regulated immobilization of Jagged1-Fc for the substrates. Mouse lineage negative cells (Lin(-)) were cultured on the Jagged1-Fc-immobilized substrates. Flow cytometric analyses demonstrated that c-Kit(+), Sca-1(+), Lin(-), and CD34(-) cells of an HSC population was significantly proliferated on the Jagged1-Fc-immobilized substrates 6 days after culture, whereas no proliferation was observed for the Jagged1-Fc-coated substrates in a random manner or Jagged1-Fc-immobilized ones with a Notch signaling inhibitor. It is concluded that the orientation-regulated immobilization of Jagged1-Fc increased the efficiency of Jagged1 to recognize the Notch receptors, resulting in the promoted ex vivo proliferation of the HSC population.

Enhancement of ectopic osteoid formation following the dual release of bone morphogenetic protein 2 and Wnt1 inducible signaling pathway protein 1 from gelatin sponges.

Bone morphogenetic protein (BMP) 2-incorporated gelatin sponge is effective for in vivo osteoinduction. However, the modeling capacity of bone decreases with age. As atrial to stimulate effective bone formation for animals with decreased osteogenic potential, Wnt1 inducible signaling pathway protein (WISP) 1, an osteoblastic regulator, was combined with gelatin sponge incorporating BMP2. Osteopontin (Opn) geneexpression was increased in vitro for mouse bone marrow stromal cells (BMSC) cultured in gelatin sponges incorporating BMP2 and WISP1 compared with those incorporating BMP2 or WISP1 alone. In vivo synergistic effect of BMP2 and WISP1 on the ectopic osteoid formation was observed when gelatin sponges incorporating BMP2 and WISP1 were implanted subcutaneously into middle-aged mice with decreased bone formation potential. It is concluded that the scaffold incorporating multiple osteoinductive agents could be effective in inducing bone formation in those with age-related decreased potential of bone formation.

Synergistic effects of the dual release of stromal cell-derived factor-1 and bone morphogenetic protein-2 from hydrogels on bone regeneration.

The objective of this study is to evaluate the activity of gelatin hydrogels incorporating combined stromal cell-derived factor-1 (SDF-1) and bone morphogenetic protein-2 (BMP-2) on the in vivo bone regeneration at an ulna critical-sized defect and subcutaneous site of rats, and compared with that of those incorporating either SDF-1 or BMP-2. The similar release profile of SDF-1 and BMP-2 from the hydrogels was observed with or without the combination of BMP-2 and SDF-1, respectively. An enhanced bone regeneration by the hydrogels incorporating combined SDF-1 and BMP-2 was observed. In addition, the implantation of hydrogels incorporating combined SDF-1 and BMP-2 enhanced the expression level of CXC chemokine cell-surface receptor-4 (Cxcr4), Runt-related factor-2 (Runx2), and Osteocalcin genes. The experiments with green fluorescent protein (GFP)-positive Chimeric mice revealed that the recruitment of bone marrow-derived cells was promoted and a vascular-like structure together with strong accumulation of CD31- and CD34-positive cells was observed at the site of hydrogels incorporating combined SDF-1 and BMP-2 implanted. In addition, a large fraction of CD29- and CD44-positive non-hematopoietic cells was detected. It is concluded that the combined release of SDF-1 and BMP-2 enhanced the recruitment of osteogenic cells and angiogenesis, resulting in the synergistic effect on bone regeneration.