Pancreatic Juice-Derived microRNA-4516 and microRNA-4674 as Novel Biomarkers for Pancreatic Ductal Adenocarcinoma.

Background and Aims: Precise diagnostic biomarkers are urgently required for pancreatic ductal adenocarcinoma (PDAC). Therefore, the aim of this study was to identify PDAC-specific exosomal microRNAs (Ex-miRs) from pancreatic juice (PJ) and evaluate their diagnostic potential. Methods: Exosomes in PJ and serum were extracted using ultracentrifugation and confirmed morphologically and biochemically. PDAC-specific Ex-miRs were identified using our original miR arrays, in which "Ex-miRs derived from the PJ of patients with chronic pancreatitis (CP)" were subtracted from Ex-miRs commonly expressed in both "human PDAC cell lines" and "the PJ of patients with PDAC." We verified the expression of these miRs using quantitative real-time reverse transcription polymerase chain reaction. Changes in serum Ex-miR levels were assessed in 2 patients with PDAC who underwent curative resection. In situ hybridization was performed to directly visualize PDAC-specific miR expression in cancer cells. Results: We identified novel Ex-miR-4516 and Ex-miR-4674 from the PJ of patients with PDAC, and they showed 80.0% and 81.8% sensitivity, 80.8% and 73.3% specificity, and 90.9% and 80.8% accuracy, respectively. The sensitivity, specificity, and accuracy of a triple assay of Ex-miR-4516/4674/PJ cytology increased to 93.3%, 81.8%, and 88.5%, respectively. In serum samples (n = 88), the sensitivity, specificity, and accuracy of Ex-miR-4516 were 97.5%, 34.3%, and 68%, respectively. Presurgical levels of serum-derived Ex-miR-4516 in 2 patients with relatively early disease stages declined after curative resection. In situ hybridization demonstrated that Ex-miR-4516 expression exclusively occurred in cancer cells. Conclusion: Liquid assays using the in situ-proven Ex-miR-4516 may have a high potential for detecting relatively early-stage PDAC and monitoring its clinical course.

Distribution, shape, and immunohistochemical characteristics of serotonin-immunoreactive neuroendocrine cells in the urethra and periurethral genital organs in mice.

The aim of this study is to clarify the disibution, shape, and immunohistochemical characteristics of serotonin-immunoreactive neuroendocrine cells (SIR-NECs) in mouse prostate and in the surrounding genital organs by histological and immunohistochemical analysis of the light microscopic serial sections of urethra. We collected lower urinary tracts from 13-week-old mice and observed the distribution pattern and shape of the SIR-NECs by serial light microscopy. The organs on the sections were divided into three anatomical zones to clarify the distribution pattern of SIR-NECs: (1) zone A, the ducts near the prostatic urethra; (2) zone B, the ducts outside the urethral sphincter; and (3) zone C, the acinus areas. Sections were double immune-stained with antibodies against serotonin and one of neuroendocrine-related factors (NRFs), including 10 neural cell markers and eight neurotransmitters, and also 4',6-diamino-2-phenylindole (DAPI). In addition, SIR-NECs were double immune-stained with antibodies against cytokeratin 5 (CK5) and p63, together with DAPI. SIR-NECs were mostly localized in zone A, and no SIR-NECs were observed in zone C. The proportion of flask-shaped SIR-NECs was approximately 15% in zones A and B. No flask-shaped SIR-NECs were observed in urethral epithelia. The NRFs co-localized with SIR-NEC were calcitonin gene-related peptide, CD56, chromogranin A, neuron-specific enolase, neuron cytoplastic protein 9.5, and synaptophysin (72.3%, 73.2%, 88.9%, 92.3%, 91.7%, and 81.9%, respectively). CK5 and p63 were not co-localized with SIR-NECs.　In this study, SIR-NEC of the urethra and the surrounding genital organs was ubiquitous in the urethra and the ducts near the urethra and co-expressed specific nerve-related NRFs.

Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM.

Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

Macular pucker formation after macular hole surgery with inverted internal limiting membrane flap technique and silicone oil tamponade.

PURPOSE: The inverted internal limiting membrane (ILM) technique was recently introduced for refractory macular hole. Here, we evaluate a case of macular pucker formation after macular hole surgery using the inverted ILM flap technique and silicone oil tamponade. After undergoing vitrectomy combined with ILM removal, the patient had a good visual prognosis. OBSERVATIONS: A 49-year-old male with macular hole affecting both of his eyes underwent vitrectomies. Three months after the first surgery in his right eye, macular pucker formation was observed in the macula, which was associated with the ILM flap used to cover the macular hole. After peeling the ILM, the macula returned to a normal contour and visual acuity improved. Examination of the removed ILM revealed macrophage-like cells containing silicone oil particles that were responsible for the ILM contraction. CONCLUSIONS AND IMPORTANCE: When using the inverted ILM flap technique and silicone oil, macular pucker may occur after macular hole surgery. Peeling of the ILM flap restored the macular shape and did not reopen the macular hole, thereby improving visual acuity. Thus, silicone oil should be used with caution when performing macular hole surgery with the ILM flap technique.

Analysis of Scars and Keloids by Focused Ion Beam/Scanning Electron Microscopy: Distinguishing Between Hypertrophic Scars and Keloids.

BACKGROUND: Histological differentiation between hypertrophic scars (HSs) and keloids has been considered difficult. In this study, we analyzed differences in the 3-dimensional tissue architecture between HSs and keloids using focused ion beam/scanning electron microscopy (FIB/SEM). METHODS: Five specimens each of normal skin, normotrophic scars (NSs), HSs, and keloids were investigated. Three sites in each specimen were observed by FIB/SEM tomography, resulting in an observation of 15 sites per tissue type. We identified fibroblasts and macrophages and assessed the contact ratio and the mode of intercellular contact (planar contact or point contact). The significance of differences among the 4 tissue types was determined by Fisher exact test. RESULTS: In normal skin, contact between fibroblasts and macrophages was observed at all 15 sites, and the mode of contact was always planar. There was contact at 87% of the NS sites (planar: point = 80%: 7%). In HSs, contact was seen at 80% of the sites (planar: point = 20%: 60%). In keloids, contact was found at only 15% of the sites (planar: point = 7.5%: 7.5%). The intercellular contact ratio showed no significant differences among normal skin, NSs, and HSs; however, a significant difference was noted between these tissues and keloids. The intercellular contact mode also showed no significant difference between normal skin and NSs, but a significant difference between these tissues and HSs. CONCLUSIONS: These histopathologic findings suggest that FIB/SEM tomography is useful for distinguishing between HSs and keloids and can provide important knowledge for understanding the pathogenesis of keloids.

Experimental study on maintaining the curvature of transplanted cartilage: Influence of the number of cartilage struts.

BACKGROUND: When auriculoplasty is performed for microtia, wire often becomes exposed during the long postoperative period. We have investigated other materials for fixing cartilage. We previously reported that absorbable sutures are more appropriate than wire. The present animal experiments investigated the reasons why fixation of transplanted cartilage is maintained when using absorbable sutures. METHODS: The costal cartilages of Sprague-Dawley rats were harvested, and three cartilage transplant models were prepared. After bending a costal cartilage into a U-shape, it was fixed by using only absorbable sutures as the control or was fixed by suturing one or two cross struts of cartilage to the U-shaped graft. Then the cartilages were subcutaneously transplanted into the backs of the rats. They were removed 8 weeks later, and the return rate of the bent cartilages was assessed. RESULTS: The return rate was 74.0%, in the suture-only group (control), 27.9% in the one-strut group, and 8.3% in the two-strut group. When the sites of contact between the U-shaped graft and the cartilage struts were observed by light microscopy, adhesion of the two cartilages by fibrous connective tissue was observed. CONCLUSION: U-shaped cartilage grafts demonstrated a smaller return rate when there was a larger contact area with the cartilage struts. Each strut was fixed by fibrous connective tissue at the contact site, thereby maintaining the shape of the graft. Thus, when creating a cartilage framework, it is important to fix the bent cartilage to the cartilage struts with a sufficiently large contact area.

Glycosylation of ascites-derived exosomal CD133: a potential prognostic biomarker in patients with advanced pancreatic cancer.

Cancer cells surviving in ascites exhibit cancer stem cell (CSC)-like features. This study analyzed the expression of the CSC marker CD133 in the ascites-derived exosomes obtained from patients with unresectable pancreatic cancer. In addition, inverse correlation of CD133 expression with prognosis was examined. Of the 133 consecutive patients, 19 patients were enrolled in the study. Exosomes derived from the malignant ascites demonstrated higher density and wider variation in size than those from non-malignant ascites. Western blot revealed enhanced expression of CD133 in exosomes obtained from patients with pancreatic cancer compared to those obtained from patients with gastric cancer or liver cirrhosis. A xenograft mouse model with malignant ascites was established by intraperitoneal inoculation of human pancreatic cancer cells in nude mice. Results obtained from the human study were reproduced in the mouse model. Statistically significant equilateral correlation was identified between the band intensity of CD133 in western blot and overall survival of patients. Lectin microarray analyses revealed glycosylation of CD133 by sialic acids as the major glycosylation among diverse others responsible for the glycosylation of exosomal CD133. These findings suggest that highly glycosylated CD133 in ascites-derived exosomes as a potential biomarker for better prognosis of patients with advanced pancreatic cancer.

Histological study of costal cartilage after transplantation and reasons for avoidance of postoperative resorption and retention of cartilage structure in rats.

BACKGROUND: Limited information is available on the biological status of transplanted cartilage from which the perichondrium has been removed. This article describes the histological and three-dimensional structural picture of cartilage, using green fluorescent protein (GFP) transgenic rats and normal wild rats. METHODS: Three sections of costal cartilage were harvested from 10-week-old wild rats. One section was used as a specimen while two were subcutaneously collected from the dorsal region of 10-week-old GFP rats at 4 and 8 weeks post-transplant. The experiment was performed in two randomized groups. The perichondrium was removed from transplanted cartilage in the first group and perichondrium of transplanted cartilage remained intact in the second group. Histology and focused ion beam/scanning electron microscope (FIB/SEM) tomography were used to evaluate the transplanted cartilage. RESULTS: All 40 transplanted sections were harvested and no infections, exposure or qualitative change of cartilage matrix were seen following transplant. Histological analyses showed that the surface layer of the GFP-negative transplanted cartilage was replaced with GFP-positive chondrocytes 8 weeks post-transplant in the first group. A three-dimensional layer of perichondrium-like tissue reconstructed around the cartilage at 8 weeks was confirmed, resembling normal perichondrium. However, the GFP-positive chondrocytes were not replaced in the second group. CONCLUSIONS: The cell renewal of chondrocytes is necessary for subcutaneously transplanted cartilage to maintain its tissue composition over a long period of time. The histological and ultrastructural analyses revealed that cells from recipient tissue generated new chondrocytes even when cartilage was implanted after removing the perichondrium.

Three-Dimensional Analysis of Peeled Internal Limiting Membrane Using Focused Ion Beam/Scanning Electron Microscopy.

PURPOSE: To reevaluate the effect of internal limiting membrane peeling during vitrectomy on the Müller cell damage, we examined the ultrastructure of the internal limiting membrane by using focused ion beam/scanning electron microscopy (FIB/SEM). METHODS: A total of 12 internal limiting membranes obtained during surgery in both the macular hole and the idiopathic epiretinal membrane groups were processed for observation by FIB/SEM. Three-dimensional structures of the internal limiting membrane were analyzed. RESULTS: The number of cell fragments in the macular hole group was 5.07 ± 1.03 per unit area of internal limiting membrane (100 µm2). The total volume of cell fragments was 3.54 ± 1.24 µm3/100 µm2. In contrast, the number of cell fragments in the epiretinal membrane group was 12.85 ± 3.45/100 µm2, and the total volume of cell fragments was 10.45 ± 2.77 µm3/100 µm2. Data for both values were significantly higher than those observed in the macular hole group (P = 0.0024 and P = 0.0022, respectively, Mann-Whitney U test). No statistical difference was found for the mean volume of the cell fragment between the two groups. CONCLUSIONS: All of the internal limiting membrane examined in this study showed cell fragments on the retinal surface of the internal limiting membrane. As compared with macular hole, epiretinal membrane exhibited a higher number and total volume of cell fragments, indicating that internal limiting membrane peeling for epiretinal membrane might have a higher risk of causing inner retinal damage. TRANSLATIONAL RELEVANCE: FIB/SEM was a useful tool for three-dimensional quantitative analysis of the internal limiting membrane.

Hyaluronic Acid Accelerates Tendon-to-Bone Healing After Rotator Cuff Repair.

BACKGROUND: There is growing evidence that the subacromial injection of hyaluronic acid (HA) is effective for pain relief in rotator cuff tears; however, its effect on tendon-to-bone healing remains unknown. PURPOSE: To examine the effect of HA on the chondrogenesis of mesenchymal stem cells (MSCs) in vitro and on tendon-to-bone healing in a rotator cuff repair model. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral complete tears of the infraspinatus tendon were made in rabbits and subsequently repaired. Before closure, 1 mL HA was applied to the repaired site, and phosphate-buffered saline was used in the opposite side as a control. Biomechanical, histological, and immunohistochemical analyses were performed at 4, 8, and 12 weeks after surgery. After euthanizing each animal, the bone marrow was isolated from the femoral bone in the same rabbits. Then, MSCs were cultured in media for chondrogenic differentiation, and the chondral pellet production and cartilage-related gene expression levels in the cells were examined at various concentrations of HA. RESULTS: At 4 and 8 weeks after surgery, ultimate load-to-failure was significantly greater in the HA group than in the control group (45.61 ± 9.0 N vs 32.42 ± 9.4 N at 4 weeks, 90.7 ± 16.0 N vs 66.97 ± 10.0 N at 8 weeks; both P < .05) but not at 12 weeks after surgery (109.6 ± 40.2 N vs 108.1 ± 42.6 N, P > .05). Linear stiffness was not significant throughout the time point evaluation. The chondroid formation area at the tendon-bone interface stained by safranin O (control vs HA group) was 0.33% ± 0.7% versus 13.5% ± 12.3% at 4 weeks after surgery ( P < .05) and 3.0% ± 5.9% versus 12.9% ± 12.9% at 8 weeks after surgery ( P < .05), but there was no significant difference at 12 weeks after surgery. Maturity of collagen at the repaired site stained by PicroSirius Red (control vs HA group) was 16.2 ± 10.6 versus 43.5 ± 21.3 at 4 weeks after surgery ( P < .05), but there were no significant differences at 8 and 12 weeks after surgery. MSCs were cultured in media for chondrogenic differentiation, and the chondral pellet production and cartilage-related gene expression levels in the cells were examined at various concentrations of HA. The number of CD44-positive cells (control vs HA group) was 8.3% ± 1.4% versus 26.2% ± 5.2% at 3 days after surgery ( P < .05), 1.8% ± 1.1% versus 26.6% ± 11.6% at 4 weeks after surgery ( P < .05), 0.6% ± 0.9% versus 0.5% ± 0.6% at 8 weeks after surgery ( P > .05), and 1.8% ± 4.0% versus 5.4% ± 4.2% at 12 weeks after surgery ( P > .05). Compared with the control group, HA significantly increased the volume of cartilaginous pellet produced by MSCs (0.0016 ± 0.0015 mm3 at 0 mg/mL of HA, 0.0041 ± 0.0023 mm3 at 1.0 mg/mL, and 0.0041 ± 0.0018 mm3 at 4.0 mg/mL), with increased mRNA expression (relative ratio to control) of type 2 collagen (1.34 ± 0.38), SOX9 (1.58 ± 0.31), and aggrecan (1.30 ± 0.22) genes in the pellet ( P < .01). CONCLUSION: HA accelerated tendon-to-bone healing in the rotator cuff repair model, enhancing the biomechanical strength and increasing chondroid formation and tendon maturity at the tendon-bone interface. Based on the data of in vitro experiments, HA-activated MSCs may play a crucial role in the acceleration of tendon-to-bone healing. CLINICAL RELEVANCE: The data suggest the relevance of clinical application of HA to accelerate tendon-to-bone healing. It may decrease the number of retears after surgery.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Histomorphometric and ultrastructural analysis of the tendon-bone interface after rotator cuff repair in a rat model.

Successful rotator cuff repair requires biological anchoring of the repaired tendon to the bone. However, the histological structure of the repaired tendon-bone interface differs from that of a normal tendon insertion. We analysed differences between the normal tendon insertion and the repaired tendon-bone interface after surgery in the mechanical properties, histomorphometric analysis, and 3-dimensional ultrastructure of the cells using a rat rotator cuff repair model. Twenty-four adult Sprague-Dawley (SD) rats underwent complete cuff tear and subsequent repair of the supraspinatus tendon. The repaired tendon-bone interface was evaluated at 4, 8, and 12 weeks after surgery. At each time point, shoulders underwent micro-computed tomography scanning and biomechanical testing (N = 6), conventional histology and histomorphometric analysis (N = 6), and ultrastructural analysis with focused ion beam/scanning electron microscope (FIB/SEM) tomography (N = 4). We demonstrated that the cellular distribution between the repaired tendon and bone at 12 weeks after surgery bore similarities to the normal tendon insertion. However, the ultrastructure of the cells at any time point had a different morphology than those of the normal tendon insertion. These morphological differences affect the healing process, partly contributing to re-tearing at the repair site. These results may facilitate future studies of the regeneration of a normal tendon insertion.

Interaction between Macrophages and Fibroblasts during Wound Healing of Burn Injuries in Rats.

Analysis of the structural changes and cell-to-cell interactions occurring during wound healing of burn injuries is essential to elucidate the morphological characteristics of the reconstitution of tissue architecture. However, conventional approaches do not provide sufficient information with respect to cell-to-cell interactions during wound healing. The aim of this study was to evaluate the interaction between bone marrow-derived cells and resident stromal cells throughout the wound healing of burn injuries, using immunohistochemistry and focused ion beam/scanning electron microscope tomography. We induced third-degree burn injuries on the backs of Wistar rats with a heated cylindrical aluminum block (2.0 cm in diameter). At 7 and 14 days after the burn injuries, the burned skin was immunostained with anti-Iba1 and anti-HSP47 antibodies for visualization of bone marrow-derived cells/macrophages and resident stromal cells/fibroblasts, respectively. Normal skin tissue was used as a control. Double-staining immunohistochemistry revealed frequent contacts between macrophages and fibroblasts and a higher contact ratio in the 3 normal skin compared with burned skin, particularly in the areas of granuloma. Three-dimensional ultrastructural analysis with focused ion beam/scanning electron microscope tomography revealed that macrophages and fibroblasts were located closer together in the normal skin than in the burned skin, confirming the analysis by light microscopic observations and ultrastructural analysis from single sections. These results highlight the importance of contact between macrophages and fibroblasts in the maintenance of skin tissue structure and during wound healing.

Effects of lidocaine on torn rotator cuff tendons.

We determined lidocaine's action on torn rotator cuff tendons in vitro and in vivo. For in vitro experiments, cell proliferation and viability assays were performed using tenocytes derived from human torn rotator cuff tendons. For in vivo experiments, acute rotator cuff tears were made on the supraspinatus tendons in the rats' bilateral shoulders; before closure, lidocaine was injected into the shoulder and saline into the contralateral shoulder (control). After sacrifice, the specimens underwent biomechanical testing or histological analysis at 24 h and at 2, 4, and 8 weeks after surgery. The extent of collagen organization and apoptosis were semi-quantitatively evaluated using collagen picrosirius red staining. Apoptosis was examined using TUNEL staining and electron microscopy. Cell proliferation decreased dose-dependently. After exposure to 0.1% lidocaine for 24 h, cell viability decreased. Two and 4 weeks after surgery, the ultimate load to failure decreased more in the lidocaine group than in the control group, with significantly reduced stiffness in the lidocaine group 2 weeks after surgery. Collagen organization significantly decreased in the lidocaine group by 4 weeks after surgery but returned to baseline at 8 weeks. TUNEL staining detected numerous apoptotic tenocytes at the torn tendon edge exposed to lidocaine 24 h after surgery; electron microscopy confirmed the condensed cell nuclei. These changes were not observed in controls. Lidocaine caused cytotoxicity to tenocytes under both conditions, decreased biomechanical properties, and induced apoptosis and delay of collagen organization in this model. Subacromial lidocaine injections in patients with rotator cuff tears should be performed carefully. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1620-1627, 2016.

Tendon-to-bone healing using autologous bone marrow-derived mesenchymal stem cells in ACL reconstruction without a tibial bone tunnel-A histological study-.

BACKGROUND: after anterior cruciate ligament (ACL) reconstruction, it is necessary to integrate free tendon graft biologically to the bone. In the present study, to verify whether a structure identical to the normal ligament-bone insertion could be regenerated at the tendon-bone interface without bone tunnel, we designed ACL reconstruction model without a tibial bone tunnel. Moreover, to enhance the integration process in this model, bone marrow-derived mesenchymal stem cells (bMSCs) were transplanted, and histological changes investigated. Our first hypothesis was that the grafted tendon would be anchored at part of the tendon-bone interface even if a bone tunnel was not created. Second hypothesis was that application of bMSCs at the tendon-bone interface would yield results histologically superior to those in controls. METHODS: bilateral ACL reconstruction using our originally designed method was performed. Autologous bMSCs with the carrier were transplanted between the bottom of the grafted tendon and the bone pit of the tibia in the experimental limb, whereas the control limb received the carrier only. At 4 and 8 weeks after the operation, histological comparison between bMSCs and the control group was carried out. RESULTS/CONCLUSIONS: even in our present ACL reconstruction model without a tibial bone tunnel, integration via chondroid tissue was seen at part of the tendon-bone interface. However, there were no appreciable differences between the groups. In ACL reconstruction, to enhance the tendon-bone integration without a bone tunnel would lead to save the graft length and prevent from bone tunnel complications (ex. Bone-tunnel enlargement after surgery).

Efficient transduction of 11 poly-arginine peptide in an ischemic lesion of mouse brain.

Direct intracellular delivery of intact proteins has been successfully achieved by tagging cell-penetrating peptide (CPP), which consists of short positively charged amino acids, such as 11 poly-arginine (11R); however, in vivo delivery of the proteins to the brain has remained challenging because it is unclear whether CPP would enable proteins to cross the blood-brain barrier (BBB). In this study, we conducted an in vivo kinetic study to investigate the efficiency of 11R-mediated peptide delivery in the normal and ischemic brain. The 11R was observed in the microvessels and neurons surrounding the microvessels throughout the brain 1 hour after systemic administration, but the signal of the peptide was faint after 2 hours. In a transient middle cerebral artery occlusion mouse model, 11R was markedly enhanced and remained detectable in the cells on the ipsilateral side for as long as 8 hours after administration compared with the contralateral side. These results suggest that 11R is capable of in vivo delivery to the brain by passing through the BBB. Furthermore, 11R-mediated protein transduction could be used for the delivery of therapeutic molecules in cerebral ischemia.

Contribution of bone marrow-derived hematopoietic stem/progenitor cells to the generation of donor-marker⁺ cardiomyocytes in vivo.

BACKGROUND: Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker(+) cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγ(null) mice. GFP(+) cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP(+) donor-derived cells, GFP(+)CFP(+) fused cells, and CFP(+) recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin(-/low)CD45(+) hematopoietic cells generated greater number of GFP(+) cardiomyocytes than Lin(-/low)CD45(-) mesenchymal cells (37.0+/-23.9 vs 0.00+/-0.00 GFP(+) cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin(-/low)Sca-1(+) or Lin(-)Sca-1(+)c-Kit(+) or CD34(-)Lin(-)Sca-1(+)c-Kit(+)) showed correlation to the number of GFP(+) cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP(+) cardiomyocytes per injected cell dose was greatest in CD34(-)Lin(-)Sca-1(+)c-Kit(+) recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP(+) cardiomyocytes than common lymphoid progenitors (12.8+/-10.7 vs 0.67+/-1.00 GFP(+) cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP(+) cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.

Bone marrow stromal cells can cause subcutaneous fibroblasts to differentiate into osteocytes in a physically stable spatial microenvironment in rats.

In this study, we investigated how rat bone marrow stromal cells (BMSCs) under a physically stable microenvironment influenced the subcutaneous fibroblasts. The model for this study involved setting up a space made up of a titanium mesh cage inserted into the subcutaneous region in rats and filled with a collagen matrix seeded with (1) BMSCs, (2) fibroblasts or (3) a combination of BMSCs and fibroblasts. Fibroblasts for transplantations were taken from enhanced green fluorescence protein (EGFP) transgenic "green rats" which enabled us to trace the fate of the cells in vivo. A series of X-ray computed tomographic (CT) images were taken of each implant over a period of 8 weeks, and the implants were then removed and examined histologically. As a result, while generated bone was observed in each case that included BMSCs (the BMSCs and combination group), there was no generated bone observed in the group using fibroblasts only. Interestingly, EGFP-positive osteocytes were observed in the generated bone of the combination group, indicating that the transplanted fibroblasts differentiated into osteocytes during the bone formation. Thus, we demonstrated that genuine intrinsic fibroblasts are able to become osteocytes as a result of the influence of BMSCs.

Feasibility of using gelatin-microbial transglutaminase complex to repair experimental retinal detachment in rabbit eyes.

BACKGROUND: To investigate the effect of using gelatin-microbial transglutaminase (gelatin-mTG) complex for treating experimental retinal detachment. METHODS: Vitrectomy with artificial posterior vitreous detachment (PVD) followed by induction of a retinal tear and detachment was performed in rabbit eyes. Gelatin-mTG complex or gelatin alone (control) was placed on the retinal tears. Fundus examination using optical coherence tomography (OCT) was performed after the surgery. Vitrectomy with PVD alone was also performed in additional rabbits. After application of the gelatin-mTG complex on the normal retinal surface, the electroretinogram (ERG) was measured 7 days after surgery. RESULTS: Gelatin-mTG complex covered the retinal tear for more than 7 days after the vitrectomy, with less prominent inflammation. Reattachment of the retina occurred in all treated eyes. In contrast, massive fibrin materials were observed at 1 day after the surgery in the control group. In addition, OCT showed that all of the gelatin disappeared by day 3. Local retinal detachment remained in three of the eyes. As demonstrated by the ERG, gelatin-mTG complex had no harmful effects on retinal function. CONCLUSIONS: The results indicate that gelatin-mTG complex continues to adhere and seal retinal tears for at least several days after administration without any inflammatory reaction.

The effect of the microenvironment created by a titanium mesh cage on subcutaneous experimental bone formation and inhibition of absorption.

We attempted to form ectopic bone under the skin of rats without adding any extrinsic bone-inducing growth factors or cytokines using bone marrow stromal cells (BMSCs), a collagen scaffold and a titanium mesh cage. We set up a space made up of a cage inserted into the subcutaneous region of rats' backs, where we could eliminate the possible influence of residual bone tissue on bone induction. We filled this space with a collagen matrix containing BMSCs. At week 8 and month 6 after implantation, the specimens were removed and observed histologically, histochemically and enzyme histochemically. As a result, bone tissue was identified in each case within the titanium cages, even though we had not used bone-inducing chemical substances. Bone generation was not found in test cases without a cage. Enhanced green fluorescence protein (EGFP) labeling of the implanted BMSCs clearly showed that these cells differentiated into osteoblasts and subsequently into osteocytes in the formed bone tissue. Host cells without EGFP labeling were also confirmed to be involved in bone formation. Six months after transplantation, the implanted cells were still present in the generated bone, and no significant resorption of the generated bone was observed. These results indicate that the physically stable spatial microenvironment created by the cage in vivo plays an important role in bone formation and inhibition of its resorption, which we refer to as the 'cage effect'.