Publications by authors named "Satoshi Fujimura"

27 Publications

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Reply: Quality and Quantity-Cultured Human Mononuclear Cells Improve Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

Plast Reconstr Surg 2021 Oct 18. Epub 2021 Oct 18.

Department of Plastic and Reconstructive Surgery Department of Regenerative Therapy Juntendo University School of Medicine Tokyo, Japan.

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http://dx.doi.org/10.1097/PRS.0000000000008545DOI Listing
October 2021

Reply: Quality and Quantity-Cultured Human Mononuclear Cells Improve Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

Plast Reconstr Surg 2021 Oct 18. Epub 2021 Oct 18.

Department of Plastic and Reconstructive Surgery Department of Regenerative Therapy Juntendo University School of Medicine Tokyo, Japan.

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http://dx.doi.org/10.1097/PRS.0000000000008543DOI Listing
October 2021

Reply: Quality and Quantity-Cultured Human Mononuclear Cells Improve the Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

Plast Reconstr Surg 2021 Oct;148(4):667e-669e

Department of Plastic and Reconstructive Surgery, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium, Departments of Plastic and Reconstructive Surgery and Regenerative Therapy, Juntendo University School of Medicine, Tokyo, Japan.

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http://dx.doi.org/10.1097/PRS.0000000000008348DOI Listing
October 2021

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

Stem Cells Transl Med 2021 Jun 18;10(6):895-909. Epub 2021 Feb 18.

Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Tokyo, Japan.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 10 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.
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http://dx.doi.org/10.1002/sctm.20-0309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133343PMC
June 2021

Quality and Quantity-Cultured Human Mononuclear Cells Improve Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

Plast Reconstr Surg 2021 02;147(2):373-385

From the Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine; and the Department of Plastic and Reconstructive Surgery, Vrije Universiteit Brussel, Universitair Ziekenhuis Brussel.

Background: Fat graft ischemia impedes us from having satisfying long-term results. The quality and quantity culture is a 1-week cell culture that increases the vasculogenic potential of peripheral blood mononuclear cells (PBMNC). This in vivo murine model investigates whether enrichment with quality and quantity-cultured human mononuclear cells (MNC-QQ) improves the vascularization in the human fat graft and whether this decreases the tissue loss.

Methods: Human adipose tissue, PBMNC, MNC-QQ, and stromal vascular fraction were prepared. First, PBMNC, MNC-QQ, and stromal vascular fraction were compared in vitro for vasculogenic potential by endothelial progenitor cell colony-forming and culture assays. Second, 0.25-g fat grafts were created with 1 × 106 PBMNC (n = 16), 1 × 106 MNC-QQ (n = 16), 1 × 106 stromal vascular fraction (n = 16), or phosphate-buffered saline as control (n = 16) before grafting in BALB/c nude mice. Grafts were analyzed for weight persistence, vessel formation by CD31 immunohistochemistry, and angiogenic markers by quantitative polymerase chain reaction.

Results: MNC-QQ develop more definitive endothelial progenitor cell colonies and more functional endothelial progenitor cells compared to PBMNC and stromal vascular fraction. Weight persistence after 7 weeks was significantly higher in grafts with MNC-QQ (89.8 ± 3.5 percent) or stromal vascular fraction (90.1 ± 4.2 percent) compared with control (70.4 ± 6.3 percent; p < 0.05). MNC-QQ-enriched grafts had the highest vessel density (96.6 ± 6.5 vessels/mm2; control, 70.4 ± 5.6 vessels/mm2; p < 0.05). MNC-QQ exerted a direct vasculogenic effect through vascular integration and a potential paracrine vascular endothelial growth factor-mediated effect.

Conclusion: Quality and quantity-cultured human mononuclear cells containing endothelial progenitor cells stimulate fat graft vascularization and enhance graft survival in a rodent recipient.
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http://dx.doi.org/10.1097/PRS.0000000000007580DOI Listing
February 2021

Changes in serum citrullinated fibrinogen concentration associated with the phase of bacteremia patients.

Clin Chim Acta 2021 Jan 4;512:127-134. Epub 2020 Nov 4.

Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan.

Background: Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase.

Methods: We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers.

Result: Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p < 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p < 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p < 0.01).

Conclusion: Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.
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http://dx.doi.org/10.1016/j.cca.2020.10.038DOI Listing
January 2021

Keloid patients have higher peripheral blood endothelial progenitor cell counts and CD34 cells with normal vasculogenic and angiogenic function that overexpress vascular endothelial growth factor and interleukin-8.

Int J Dermatol 2019 Dec 9;58(12):1398-1405. Epub 2019 Jul 9.

Department of Plastic, Reconstructive and Aesthetic Surgery, Nippon Medical School, Tokyo, Japan.

Background: One suggested reason for aberrant wound healing in keloid scars is chronic inflammation of the dermis. We hypothesized that excessive blood vessel formation and high capillary density in keloid tissue is caused by dysfunction of endothelial progenitor cells.

Methods: We compared the number of circulating endothelial progenitor cells and vasculogenic and angiogenic capacity, as well as secretory function, of circulating CD34 cells in keloid patients and healthy individuals.

Results: Compared to mononuclear cell cultures from healthy donors, cultures of peripheral blood mononuclear cells obtained from keloid patients showed a more than twofold increase in the number of peripheral blood EPCs (fibronectin-adhering cells that phagocytized acetylated low-density lipoprotein and bound Ulex europaeus agglutinin-I lectin). However, there was no difference in colony-forming ability and participation in in vitro angiogenesis between circulating CD34 cells isolated from keloid patients and healthy individuals. This means that circulating CD34 /endothelial progenitor cells in keloid patients have normal vasculogenic and angiogenic function. However, CD34 cells derived from keloid patients demonstrated a more than sevenfold expression of the interleukin-8 gene and a more than fivefold expression of the vascular endothelial growth factor gene than CD34 cells derived from healthy individuals.

Conclusions: These results support the role of vascular endothelial growth factor and interleukin-8 in increased recruitment of endothelial progenitor cells in keloid patients.
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http://dx.doi.org/10.1111/ijd.14575DOI Listing
December 2019

Quality and Quantity-Cultured Murine Endothelial Progenitor Cells Increase Vascularization and Decrease Fibrosis in the Fat Graft.

Plast Reconstr Surg 2019 Apr;143(4):744e-755e

From the Plastic and Reconstructive Surgery Department, Juntendo University School of Medicine; and the Plastic and Reconstructive Surgery Department, Universitair Ziekenhuis Brussel (University Hospital Brussels), Vrije Universiteit Brussel.

Background: Fat grafting has become a valuable technique for soft-tissue reconstruction; however, long-lasting success depends on several determinants. An early blood supply to the transplanted adipocytes is important to prevent ischemia. The recently developed quality and quantity (QQ) culture increases the vasculogenic potential of endothelial progenitor cells. The authors used a murine fat grafting model to address the hypothesis that QQ-cultured endothelial progenitor cells stimulate the establishment of a blood vessel network and increase graft success.

Methods: c-KitSca-1Lin (KSL) cells were isolated as endothelial progenitor cell precursors from C57BL/6 mice. Adipose tissue was grafted with QQ-cultured KSL cells (QQKSL group), uncultured KSL cells (KSL group), adipose-derived stem cells (ASC group), and a combination (QQKSL+ASC group), and compared to a control group. Five and 10 weeks later, grafts were weighed, histologic and immunohistochemical parameters were evaluated, and gene expression was quantified by quantitative polymerase chain reaction.

Results: The highest vessel density was observed in the combined QQKSL+ASC group (68.0 ± 4.3/mm; p < 0.001) and the QQKSL group (53.9 ± 3.0/mm; p < 0.001). QQKSL cells were engrafted in proximity to the graft vasculature. QQKSL cells decreased the fibrosis percentage (13.8 ± 1.8 percent; p < 0.05). The combined QQKSL+ASC group (22.4 ± 1.8/mm; p < 0.001) showed the fewest local inflammation units. A significant up-regulation of platelet-derived growth factor and adiponectin expression was observed in the QQKSL group and QQKSL+ASC group. Graft weight persistence was not significantly different between groups.

Conclusions: Supplementing fat grafts with quality and quantity-cultured endothelial progenitor cells improves graft quality by stimulating vascularization. The increased vessel density is associated with less fibrosis, less inflammation, and better adipose tissue integrity. Enriching fat grafts with QQ-cultured endothelial progenitor cells is a potential solution to their clinical shortcomings.
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http://dx.doi.org/10.1097/PRS.0000000000005439DOI Listing
April 2019

Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model.

Cell Transplant 2018 07 5;27(7):1068-1079. Epub 2018 Jul 5.

1 Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Tokyo, Japan.

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 10 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34 and CD133 stem cell populations, significant reduction in CCR2 cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206 cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.
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http://dx.doi.org/10.1177/0963689718780307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158547PMC
July 2018

Quality-Quantity Control Culture Enhances Vasculogenesis and Wound Healing Efficacy of Human Diabetic Peripheral Blood CD34+ Cells.

Stem Cells Transl Med 2018 05 24;7(5):428-438. Epub 2018 Mar 24.

Department of Basic Clinical Science, Division of Regenerative Medicine, Tokai University School of Medicine, Isehara, Japan.

Autologous endothelial progenitor cell (EPC) therapy is commonly used to stimulate angiogenesis in ischemic repair and wound healing. However, low total numbers and functional deficits of EPCs make autologous EPC therapy ineffective in diabetes. Currently, no known ex vivo culture techniques can expand and/or ameliorate the functional deficits of EPCs for clinical usage. Recently, we showed that a quality-quantity culture (QQc) system restores the vasculogenic and wound-healing efficacy of murine diabetic EPCs. To validate these results and elucidate the mechanism in a translational study, we evaluated the efficacy of this QQc system to restore the vasculogenic potential of diabetic human peripheral blood (PB) CD34+ cells. CD34+ cells purified from PB of diabetic and healthy patients were subjected to QQc. Gene expression, vascular regeneration, and expression of cytokines and paracrine mediators were analyzed. Pre- or post-QQc diabetic human PB-CD34+ cells were transplanted into wounded BALB/c nude mice and streptozotocin-induced diabetic mice to assess functional efficacy. Post-QQc diabetic human PB-CD34+ cell therapy significantly accelerated wound closure, re-epithelialization, and angiogenesis. The higher therapeutic efficacy of post-QQc diabetic human PB-CD34+ cells was attributed to increased differentiation ability of diabetic CD34+ cells, direct vasculogenesis, and enhanced expression of angiogenic factors and wound-healing genes. Thus, QQc can significantly enhance the therapeutic efficacy of human PB-CD34+ cells in diabetic wounds, overcoming the inherent limitation of autologous cell therapy in diabetic patients, and could be useful for treatment of not only wounds but also other ischemic diseases. Stem Cells Translational Medicine 2018;7:428-438.
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http://dx.doi.org/10.1002/sctm.17-0043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905232PMC
May 2018

The role of Notch signaling in diabetic endothelial progenitor cells dysfunction.

J Diabetes Complications 2016 Jan-Feb;30(1):12-20. Epub 2015 Sep 30.

Department of Cardiovascular Medicine, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, 113-8421, Japan. Electronic address:

Aims: To investigate the role of Notch signaling pathway in vasculogenic dysfunction of diabetic EPCs (DM-EPCs).

Methods: The study was performed in mice and diabetes was induced with Streptozotocin. The functional consequences of Notch pathway modulation were studied by assessment of colony forming capacity (EPC colony forming assay), EPC differentiation capacity (% of definitive EPC-CFU (dEPC-CFU)), circulating EPCs (EPC culture assay) and migrated cells (migration assay); in the presence of Notch inhibitor (γ-secretase inhibitors (GSI)) compared to control. Notch pathway and VEGF involvement in DM- EPCs were assessed by gene expression (RT-qPCR).

Results: DM demonstrated to increase Notch pathway expression in bone marrow (BM) EPCs followed by lower EPC-CFU number, EPCs differentiation capacity, number of circulating EPCs, migrated cells and VEGF expression compared to control (p<0.05). Inhibition of Notch pathway by GSI rescued vasculogenic dysfunction in DM-EPCs as represented by increase in EPC-CFU number, differentiation capacity and number of circulating EPCs (p<0.05).

Conclusion: Our findings indicate the involvement of Notch pathway in mediating DM-EPCs dysfunction including less number of EPC-CFU, circulating EPCs and migrated cell number compared to control. Further in vitro inhibition of Notch pathway by GSI rescued DM-EPC dysfunction. Therefore targeting Notch pathway in DM may provide a target to restore DM-EPC dysfunction.
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http://dx.doi.org/10.1016/j.jdiacomp.2015.09.015DOI Listing
October 2016

Oxidative stress tolerance of early stage diabetic endothelial progenitor cell.

Regen Ther 2015 Jun 23;1:38-44. Epub 2015 Feb 23.

Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

Introduction: One of the causes for poor vasculogenesis of diabetes mellitus (DM) is known to rise from the dysfunction of bone marrow-derived endothelial progenitor cells (BM EPCs). However, the origin of its cause is less understood. We aimed to investigate the effect of oxidative stress in early stage of diabetic BM-EPC and whether its vasculogenic dysfunction is caused by oxidative stress.

Methods: Bone marrow c-Kit+Sca-1+Lin- (BM-KSL) cells were sorted from control and streptozotocin-induced diabetic C57BL6J mice by flow cytometry. BM-KSLs were then assessed for vasculogenic potential (colony forming assay; EPC-CFA), accumulation of intracellular ROS (CM-H2DCFDA), carbonylated protein (ELISA), anti-oxidative enzymes expression (RT-qPCR) and catalase activity (Amplex Red).

Results: Compared to control, DM BM-KSL had significantly lower EPC-CFUs in both definitive EPC-CFU and total EPC-CFU (p < 0.05). Interestingly, the oxidative stress level of DM BM-KSL was comparable and was not significantly different to control followed by increased in anti-oxidative enzymes expression and catalase activity.

Conclusions: Primitive BM-EPCs showed vasculogenic dysfunction in early diabetes. However the oxidative stress is not denoted as the major initiating factor of its cause. Our results suggest that primitive BM-KSL cell has the ability to compensate oxidative stress levels in early diabetes by increasing the expression of anti-oxidative enzymes.
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http://dx.doi.org/10.1016/j.reth.2014.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581786PMC
June 2015

Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential.

J Am Heart Assoc 2014 Jun 25;3(3):e000743. Epub 2014 Jun 25.

Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan (H.M., T.S., A.S., T.A.) Vascular Regeneration Research Group, Institute of Biomedical Research and Innovation IBRI, Kobe, Japan (H.A., A.K., T.A.).

Background: Cell-based therapies involving mononuclear cells (MNCs) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNCs has not been evaluated. We investigated the therapeutic potential of peripheral blood (PB) MNCs, operated by recently developed quality and quantity (QQ) culture of endothelial progenitor cells (EPCs).

Methods And Results: PBs were collected from healthy volunteers; peripheral blood mononuclear cells (PBMNCs) isolated from these PBs were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6. The resulting cells (QQMNCs) in EPC colony-forming assay generated significantly more definitive EPC colonies than PBMNCs. In flow cytometry, macrophages and helper T lymphocytes of QQMNCs became phenotypically polarized into angiogenic, anti-inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T-cell expansion. Quantitative real-time polymerase chain reaction (qRT-PCR) assay revealed the predominant proangiogenic gene expressions in QQMNCs versus PBMNCs. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured "early EPC" Tx (eEPCTx), and granulocyte colony-stimulating factor mobilized CD34(+) cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx. Histological evaluations and qRT-PCR assays in ischemic hindlimbs demonstrated that QQMNCTx, similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx.

Conclusions: QQ culture potentiates the ability of PBMNCs to promote regeneration of injured tissue; considering the feasible cell preparation, QQ culture-treated PBMNCs may provide a promising therapeutic option for ischemic diseases.

Clinical Trial Registration Url: irb.med.u-tokai.ac.jp/d/2/monthly/2010.html; IRB No.: 10R-020.URL: irb.med.u-tokai.ac.jp/d/2/monthly/201312.html; IRB No.: 13R228.
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http://dx.doi.org/10.1161/JAHA.113.000743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309104PMC
June 2014

Evaluation of bioartificial renal tubule device prepared with human renal proximal tubular epithelial cells cultured in serum-free medium.

J Artif Organs 2013 Sep 12;16(3):368-75. Epub 2013 May 12.

Division of Nephrology and Metabolism, Department of Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.

Bioartificial renal tubule devices (BTD) use cell therapy to improve conditions commonly observed in recipients of artificial kidneys for treatment of kidney diseases. We previously reported significant improvement of the condition of acute kidney injury (AKI) animals after treatment with BTD prepared with lifespan-extended human renal proximal tubular cells (hRPTEC). However, a major obstacle to use of BTD for patients is their biological safety, because hRPTEC are cultured in medium containing fetal calf serum. To establish the biological safety of BTD, we prepared BTD with lifespan-extended hRPTEC cultured in a newly developed serum-free medium and compared these with BTD prepared with hRPTEC cultured in serum-containing conventional medium. Lifespan-extended hRPTEC cultured in serum-free medium (hRPTEC-SFM) can proliferate similar to hRPTEC cultured in serum-containing conventional medium (hRPTEC-CM). Comparison of leakage and of reabsorption of small molecules for BTD prepared with hRPTEC-SFM (BTD-SFM) with those for our previous BTD prepared with hRPTEC-CM (BTD-CM) showed transportation in these two types of BTD was almost identical. When AKI goats were treated with BTD-SFM for 26 h, increase of survival time and reduction of cytokine expression in blood cells were almost same as for AKI goats treated with BTD-CM. Quantification of the expression of some genes of hRPTEC in BTD revealed significant changes during BTD treatment for AKI goats. In conclusion, lifespan-extended hRPTEC-SFM work as well as hRPTEC-CM, and the biological safety of BTD for patients could be elevated without loss of function by preparation from hRPTEC-SFM.
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http://dx.doi.org/10.1007/s10047-013-0710-8DOI Listing
September 2013

Anagliptin, a DPP-4 inhibitor, suppresses proliferation of vascular smooth muscles and monocyte inflammatory reaction and attenuates atherosclerosis in male apo E-deficient mice.

Endocrinology 2013 Mar 21;154(3):1260-70. Epub 2013 Jan 21.

Department of Metabolism & Endocrinology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

Dipeptyl peptidase-4 (DPP-4) inhibitors modulate the progression of atherosclerosis. To gain insights into their mechanism of action, 9-wk-old male apolipoprotein E (apoE)-deficient mice were fed a DPP-4 inhibitor, anagliptin-containing diet. The effects of anagliptin were investigated in, a monocyte cell line, human THP-1 cells, and rat smooth muscle cells (SMCs). Treatment with anagliptin for 16 wk significantly reduced accumulation of monocytes and macrophages in the vascular wall, SMC content in plaque areas, and oil red O-stained area around the aortic valve without affecting glucose tolerance or body weight. Serum DPP-4 concentrations were significantly higher in apoE-deficient mice than control mice, and the levels increased with aging, suggesting the involvement of DPP-4 in the progression of atherosclerosis. Indeed, soluble DPP-4 augmented cultured SMC proliferation, and anagliptin suppressed the proliferation by inhibiting ERK phosphorylation. In THP-1 cells, anagliptin reduced lipopolysaccharide-induced TNF-α production with inhibiting ERK phosphorylation and nuclear translocation of nuclear factor-κB. Quantitative analysis also showed that anagliptin reduced the area of atherosclerotic lesion in apoE-deficient mice. These results indicated that the anti-atherosclerotic effect of anagliptin is mediated, at least in part, through its direct inhibition of SMC proliferation and inflammatory reaction of monocytes.
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http://dx.doi.org/10.1210/en.2012-1855DOI Listing
March 2013

Evaluation of bioartificial renal tubule device prepared with lifespan-extended human renal proximal tubular epithelial cells.

Nephrol Dial Transplant 2012 Aug 30;27(8):3091-9. Epub 2012 Mar 30.

Division of Nephrology and Metabolism, Department of Medicine, Tokai University School of Medicine, Isehara, Japan.

Background: Acute kidney injury (AKI), accompanied by the development of systemic inflammatory response syndrome and multiorgan dysfunction syndrome, is associated with a high risk of death. Bioartificial renal tubule device (BTD) is a cell therapy that improves the conditions common to artificial kidney recipients treated for kidney diseases. In this paper, we describe the establishment of BTD with lifespan-extended human renal proximal tubular epithelial cells.

Methods: AKI goats were established by performing bilateral nephrectomy followed by lipopolysaccharide administration. The AKI goats were treated with BTD or sham-BTD, and the two groups of animals were compared by measuring the respective life spans and the levels of blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and serum electrolytes. The expression levels of inflammatory cytokines were detected by reverse transcription-polymerase chain reaction, and plasma interleukin (IL)-6 levels were measured by enzyme-linked immunosorbent assay.

Results: The life span of AKI goats was extended: the lifetime with the BTD treatment compared with sham-BTD. BTD and sham-BTD showed a similar degree of small solute clearance. The expression levels of inflammatory cytokines and plasma IL-6 levels were decreased by the BTD treatment.

Conclusions: BTD treatment results in less damage from endotoxin shock and increased life span in AKI goats. These results suggest that BTD may be a useful component of bioartificial kidneys and should be considered in the next generation of renal replacement therapies.
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http://dx.doi.org/10.1093/ndt/gfr755DOI Listing
August 2012

Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular epithelial cells.

Nephrol Dial Transplant 2011 Sep 18;26(9):2761-9. Epub 2011 Mar 18.

Division of Nephrology and Metabolism, Department of Medicine, Tokai University School of Medicine, Isehara, Japan.

Background: The bioartificial renal tubule device is a cell therapy system for renal failure. The major obstacle in the development of the bioartificial renal tubule device is the obtainment of a large number of viable renal tubule cells to seed on the inner surface of hollow fibers. Although our previous studies had used a transformed cell line, they may be dangerous for clinical uses. Therefore, different approaches to amplify renal proximal tubular epithelial cells (RPTEC) in culture without oncogenes, vectors and carcinogens have been required.

Methods: The limitation of the replicative lifespan of human RPTEC, which is ∼12 population doublings (PDs), was extended by invalidating messenger RNA of cell cycle-related genes with antisense oligonucleotide or small interfering RNA (siRNA).

Results: Periodic transfection of siRNA to a tumor suppressor p53 or a cyclin-dependent kinase inhibitor p16(INK4a) extended the lifespan by 33 and 63 PDs, respectively, in 3 months of culture. The siRNA-mediated lifespan extension was controllable because cell division ceased within 2 weeks after the transfection was discontinued. Expressions of γ-glutamyltransferase 1 and glucose transporter 1 were recovered in siRNA-transfected RPTEC cultured on porous membranes. Bioartificial renal tubule devices (0.8 m(2)) constructed with these cells showed reabsorption of water (122.3 ± 4.2 mL/30 min), sodium (18.1 ± 0.7 mEq/30 min) and glucose (121.7 ± 4.4 mg/30 min) after 1 week of circulation. Furthermore, β2-microglobulin and pentosidine were metabolized by RPTEC in mini-devices (65 cm(2)) within 48 h of circulation.

Conclusions: These approaches enabled us to yield a high enough number of RPTEC for construction of bioartificial renal tubule devices repeatedly. Lifespan-extended RPTEC could recover their specific characteristics by culturing on porous membranes, and bioartificial renal tubule devices constructed with these cells showed good performances of reabsorption and metabolism.

Summary: A large number of human renal tubular cells required for construction of the bioartificial renal tubule device were prepared by extending the lifespan of the primary cells by invalidating mRNA of cell cycle-related genes. Constructed bioartificial renal tubule devices with lifespan-extended cells showed good performances of in vitro examination of reabsorption and metabolism. Requiring no oncogenes, vectors or cell cloning, the RNAi-mediated lifespan extension can help advance tissue-replacement therapy as well as basic research.
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http://dx.doi.org/10.1093/ndt/gfr066DOI Listing
September 2011

Present status and future perspectives on the development of bioartificial kidneys for the treatment of acute and chronic renal failure patients.

Hemodial Int 2011 Apr 13;15(2):183-92. Epub 2011 Mar 13.

Department of Medicine, Division of Nephrology and Metabolism, Tokai University School of Medicine, Isehara, Japan.

A bioartificial renal tubule device (BTD) consisting of a hollow-fiber module and human proximal tubular epithelial cells has been completed technically by Humes and colleagues and a few other groups. Humes and colleagues developed BTD, treated acute kidney injury patients with multiorgan failure by continuous hemofiltration (CHF) in conjunction with BTD, and reported a significantly higher survival rate than that by CHF with BTD without cells in the Food and Drug Administration phase IIa trial. However, BTD has never been approved by the US Government, as the CHF+BTD treatment did not show a significant difference from the control group in the phase IIb trial. Human proximal tubular epithelial cells were confirmed to be overgrown on artificial membrane, which resulted in the inhibition of active transports and the metabolism of essential substances. Function of the BTD could be maintained in a U0126-contained medium, even if the BTD had to have been waited by a new acute kidney injury patient for several weeks. For wearable kidneys, heparin-covalently bound membrane or methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated membranes are candidates for antithrombogenic hemofilters, while endothelial progenitor cells from a cord blood, CD133(+) cells-attached hemofilter in which the permeability of the cells was enhanced by the enlarged diameter of fenestrae by treating with cytochalasin B are another candidate. The MPC blend membrane containing 1% of the MPC polymer in polysulfone was developed as a BTD module. MPC was 7 times larger at the sponge layer than at the skin layer of the membrane, resulting in hemocompatibility at the sponge layer and cytocompatibility at the skin layer.
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http://dx.doi.org/10.1111/j.1542-4758.2011.00530.xDOI Listing
April 2011

CD133+ endothelial progenitor cells as a potential cell source for a bioartificial glomerulus.

Tissue Eng Part A 2009 Oct;15(10):3173-82

Division of Nephrology and Metabolism, Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan.

Development of a bioartificial glomerulus, a hemofilter in which the inner surface of hollow fibers is endothelialized, requires expandable, nonimmunogenic, antithrombogenic, and highly permeable endothelial cells. We used human umbilical cord blood CD133(+) endothelial progenitor cells (EPCs) to evaluate the feasibility of application of EPCs for bioartificial glomerulus. Numbers of adhered CD133(+) EPCs (adhered EPCs) was approximately 25 to 30 times as great in the expansion culture group as in the non-expansion group. Adhered EPCs had endothelial cell features, including the expression of CD31, Kinase domain region, von Willebrand factor, vascular endothelial-cadherin, positive for Ulex europeus agglutinin I staining, and up-take of acetylated low-density lipoprotein. Adhered EPCs secreted 6-keto-prostaglandin F(1alpha) identically to that secreted by human umbilical vein endothelial cells (HUVECs). The cells also expressed messenger RNA for phospholipase A(2), cyclooxygenase (COX)-1, COX-2, prostaglandin I(2) synthase, tissue plasminogen activator, and thrombomodulin (TM). TM protein in adhered EPCs properly activated protein C. Scanning electron microscopy revealed the suppression of platelet adhesion and aggregation on the surface of cell monolayer. Adhered EPCs treated with 50 microg/mL of cytochalasin B induced a larger diameter and a greater number of fenestrae, subsequently producing significantly more ultrafiltration than the non-treated cell. These results suggest that CD133(+) EPCs would potentially be applicable in bioartificial glomerulus.
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http://dx.doi.org/10.1089/ten.TEA.2009.0050DOI Listing
October 2009

Hunter-killer peptide (HKP) for targeted therapy.

J Med Chem 2008 Oct;51(19):5887-92

College of Pharmacy, Touro University, Mare Island, Vallejo, California 94592, USA.

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http://dx.doi.org/10.1021/jm800495uDOI Listing
October 2008

Characterization of mouse striatal precursor cell lines expressing functional dopamine receptors.

Dev Neurosci 2006 ;28(6):518-27

Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Dopamine and its receptors appear in the developing brain early in the embryonic period and dopamine receptor activation influences proliferation and differentiation of neuroepithelial precursor cells. Since dopamine D(1) and D(2) receptor activation produces opposing effects on precursor cell activity, dopamine's overall effects may correlate with relative numbers and activity of each receptor subtype on the precursor cells. Dopamine receptor expression and activity in individual precursor cells in the intact brain are difficult to ascertain. Therefore, cell lines with known receptor expression profiles can be useful tools to study dopamine's influence on neuroepithelial cells. We report characterization of dopamine receptor expression and activity profiles in three mouse striatal precursor cell lines and suggest that these cell lines can be valuable tools to study dopamine's effects on striatal precursor cell proliferation and differentiation.
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http://dx.doi.org/10.1159/000095114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1615710PMC
November 2006

Structural evaluation of a novel pro-apoptotic peptide coupled to CNGRC tumor homing sequence by NMR.

Chem Biol Drug Des 2006 Jun;67(6):417-24

Chemistry Department, University of San Diego, San Diego, CA 92110, USA.

Hunter-killer peptides (HKPs) are synthetic peptides that target specific cell types for apoptosis. These studies report functional and structural characteristics of HKP9, an hunter-killer peptide that specifically targets tumor vasculature with a new apoptotic sequence. Vesicle leakage experiments were performed as a model for membrane perturbing activity. Placement of the homing sequence reduces both cell toxicity and vesicle leakage activity. NMR studies elucidate the conformation and orientation of HKP9 in micelles. The positively charged end of the HKP9 killing sequence is solvent exposed; however, the central portion of the peptide is helical and buried in dodecylphosphorylcholine micelles. The homing sequence is less solvent exposed than in a previously reported tumor-homing peptide. The results suggest that solvent accessibility of the homing sequence should be considered in design of future peptides.
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http://dx.doi.org/10.1111/j.1747-0285.2006.00394.xDOI Listing
June 2006

Growth arrest and apoptosis in adult T cell leukemia cell lines following IL-2 deprivation.

Int J Oncol 2004 Aug;25(2):437-43

Molecular Oncology Center, Department of Laboratory Medicine, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan.

Adult T-cell leukemia (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type-I (HTLV-I). Since ATL cells often require IL-2 for their proliferation and survival, we examined the effect of IL-2 deprivation on the IL-2-dependent ATL cells established from ATL patients. After IL-2 withdrawal, these cells were arrested in the G1 phase and then underwent apoptosis. p27Kip1 was observed to act as a cell cycle inhibitor. A decrease in the amount of Bcl-xL was more distinct than that of Bcl-2, while Bax increased slightly during IL-2 withdrawal. The activation of caspase-3 and the loss of mitochondrial membrane potential were also observed. An overexpression of Bcl-xL protein in the KK1, one of the ATL cell lines, suppressed apoptosis by the 3rd day, however, apoptosis could not be prevented completely. Thereafter, a decrease in Bcl-xL and an activation of caspase-3 were observed even under the overexpression of Bcl-xL. The mitochondrial membrane potential and the intra-cellular levels of reactive oxygen species (ROS) also changed due to IL-2 deprivation. From these results, the IL-2 signals are considered to be essential for the survival of ATL cells, and the interruption of IL-2 signaling might thus be useful as a potentially new treatment for ATL.
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August 2004

Islet regeneration during the reversal of autoimmune diabetes in NOD mice.

Science 2003 Nov;302(5648):1223-7

Immunobiology Laboratory, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Room 3602, Charlestown, MA 02129, USA.

Nonobese diabetic (NOD) mice are a model for type 1 diabetes in humans. Treatment of NOD mice with end-stage disease by injection of donor splenocytes and complete Freund's adjuvant eliminates autoimmunity and permanently restores normoglycemia. The return of endogenous insulin secretion is accompanied by the reappearance of pancreatic beta cells. We now show that live donor male or labeled splenocytes administered to diabetic NOD females contain cells that rapidly differentiate into islet and ductal epithelial cells within the pancreas. Treatment with irradiated splenocytes is also followed by islet regeneration, but at a slower rate. The islets generated in both instances are persistent, functional, and apparent in all NOD hosts with permanent disease reversal.
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http://dx.doi.org/10.1126/science.1088949DOI Listing
November 2003

Downregulation of Bcl-xL and activation of caspases during retinoic acid-induced apoptosis in an adult T-cell leukemia cell line.

Hematol J 2003 ;4(5):328-35

Molecular Oncology Center, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka, Japan.

Introduction: Adult T-cell leukemia (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type I (HTLV-I). We previously reported that retinoic acid (RA) isomers, all-trans-RA (ATRA), 9-cis-RA and 13-cis-RA, induce growth arrest and apoptosis in ATL cell lines. In this study we investigate the mechanisms of apoptosis induced by RAs.

Materials And Methods: An ATL cell line, KK1, established from an ATL patient was cultured with or without RAs. DNA fragmentation was analyzed using propidium iodide for staining DNA on a FACS. The apoptosis-related proteins and their transcriptional levels were thereafter analyzed by Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Caspase activity was determined by using specific fluorogenic substrates.

Results: Bcl-2 and Bcl-xL are constitutively expressed in KK1. Treatment with RAs caused the level of Bcl-x protein decreased, while the levels of Bcl-2 and Bcl-xS remained unchanged. The level of Bax tended to decrease. There was a reduction of mitochondrial membrane potential and activation of caspase-3 and -6 without any activation of caspase-1. Broad range caspase inhibitors, Z-Asp and Z-VAD, prevented DNA fragmentation.

Conclusion: These results suggest that the RA-induced apoptotic signals were transduced via downregulation of Bcl-xL and the decrease in the mitochondrial membrane function leading to caspase-3 activation.
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http://dx.doi.org/10.1038/sj.thj.6200321DOI Listing
June 2004

Tumor growth suppression by a retroviral vector displaying scFv antibody to CEA and carrying the iNOS gene.

Anticancer Res 2002 Jul-Aug;22(4):2443-6

First Department of Biochemistry, Fukuoka University School of Medicine, Japan.

Background: In a recent in vitro study we demonstrated a specifically-targeted killing of CEA-expressing cells by a recombinant bifunctional retrovector displaying an scFv antibody to CEA and carrying the iNOS gene. In this study, we tested whether a gene therapy using the recombinant retrovirus could inhibit the growth of CEA-expressing tumors in mice.

Materials And Methods: SCID mice were inoculated s.c. on the back with CEA-expressing MKN-45 cells on day 0. The recombinant viral particles were injected into the inoculated sites on days 3, 5 and 7 and tumor size was measured every 5 days.

Results: The s.c. administration of the recombinant retrovirus produced a marked growth inhibition of MKN-45 tumors in SCID mice. When the actual tumor weights were measured 50 days after initiation of treatment, about 70% reduction was observed in the treated group as compared to the control groups.

Conclusion: This approach may also be applied to other tumor antigens expressed on cancer cells and is a step towards the cell specific suppression of tumorigenicity.
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September 2002
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