Koji Kojima

Stimulus-triggered fate conversion of somatic cells into pluripotency

Here we report a unique cellular reprogramming phenomenon, called stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors. In STAP, strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells. Through real-time imaging of STAP cells derived from purified lymphocytes, as well as gene rearrangement analysis, we found that committed somatic cells give rise to STAP cells by reprogramming rather than selection. STAP cells showed a substantial decrease in DNA methylation in the regulatory regions of pluripotency marker genes. Blastocyst injection showed that STAP cells efficiently contribute to chimaeric embryos and to offspring via germline transmission. We also demonstrate the derivation of robustly expandable pluripotent cell lines from STAP cells. Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

Study Design. By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. Objectives. The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. Summary of Background Data. Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. Methods. Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in −70 C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. Results. The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. Conclusion. These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Hams F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell‐polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudostratified columnar epithelium, with a distinct interface between tissue‐engineered cartilage and epithelium. Safranin‐O staining shows that tissue‐engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.—Kojima, K., Bonassar, L. J., Roy, A. K., Mizuno, H., Cortiella, J., Vacanti, C. A. A composite tissue‐engineered trachea using sheep nasal chondrocyte and epithelial cells. FASEB J. 17, 823–828 (2003)

Bioengineered Three-Layered Robust and Elastic Artery Using Hemodynamically-Equivalent Pulsatile Bioreactor

Background— There is an essential demand for tissue engineered autologous small-diameter vascular graft, which can function in arterial high pressure and flow circulation. We investigated the potential to engineer a three-layered robust and elastic artery using a novel hemodynamically-equivalent pulsatile bioreactor. Methods and Results— Endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were harvested from bovine aorta. A polyglycolic acid (PGA) sheet and a polycaprolactone sheet seeded with SMCs, and a PGA sheet seeded with fibroblast, were wrapped in turn on a 6-mm diameter silicone tube and incubated in culture medium for 30 days. The supporting tube was removed, and the lumen was seeded with ECs and incubated for another 2 days. The pulsatile bioreactor culture, under regulated gradual increase in flow and pressure from 0.2 (0.5/0) L/min and 20 (40/15) mm Hg to 0.6 (1.4/0.2) L/min and 100 (120/80) mm Hg, was performed for an additional 2 weeks (n=10). The engineered vessels acquired distinctly similar appearance and elasticity as native arteries. Scanning electron microscopic examination and Von Willebrand factor staining demonstrated the presence of ECs spread over the lumen. Elastica Van Gieson and Masson Tricrome Stain revealed ample production of elastin and collagen in the engineered grafts. Alpha-SMA and calponin staining showed the presence of SMCs. Tensile tests demonstrated that engineered vessels acquired equivalent ultimate strength and similar elastic characteristics as native arteries (Ultimate Strength of Native: 882±133 kPa, Engineered: 827±155 kPa, each n=8). Conclusions— A robust and elastic small-diameter artery was engineered from three types of vascular cells using the physiological pulsatile bioreactor.

In Vitro Tissue Engineering to Generate a Human-Sized Auricle and Nasal Tip

Objectives/Hypothesis Tissue engineering has successfully generated cartilage in a xenograft and an autograft model. However, challenges remain with both of these in vivo techniques before clinical application can be realized. We hypothesized that a human‐sized cartilaginous structure could be generated completely in vitro as a complementary or an alternative technique.

Isoflurane Decreases Self-Renewal Capacity of Rat Cultured Neural Stem Cells

Background: In models, isoflurane produces neural and behavioral deficits in vitro and in vivo. This study tested the hypothesis that neural stem cells are adversely affected by isoflurane such that it inhibits proliferation and kills these cells. Methods: Sprague-Dawley rat embryonic neural stem cells were plated onto 96-well plates and treated with isoflurane, 0.7, 1.4, or 2.8%, in 21% oxygen for 6 h and fixed either at the end of treatment or 6 or 24 h later. Control plates received 21% oxygen under identical conditions. Cell proliferation was assessed immunocytochemically using 5-ethynyl-2′-deoxyuridine incorporation and death by propidium iodide staining, lactate dehydrogenase release, and nuclear expression of cleaved caspase 3. Data were analyzed at each concentration using an ANOVA; P < 0.05 was considered significant. Results: Isoflurane did not kill neural stem cells by any measure at any time. Isoflurane, 1.4 and 2.8%, reduced cell proliferation based upon 5-ethynyl-2′-deoxyuridine incorporation, whereas isoflurane, 0.7%, had no effect. At 24 h after treatment, the net effect was a 20–30% decrease in the number of cells in culture. Conclusions: Isoflurane does not kill neural stem cells in vitro. At concentrations at and above the minimum alveolar concentrations required for general anesthesia (1.4 and 2.8%), isoflurane inhibits proliferation of these cells but has no such effect at a subminimum alveolar concentration (0.7%). These data imply that dosages of isoflurane at and above minimum alveolar concentrations may reduce the pool of neural stem cells in vivo but that lower dosages may be devoid of such effects.

Comparison of Tracheal and Nasal Chondrocytes for Tissue Engineering of the Trachea

BACKGROUND This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage-derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. METHODS Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. RESULTS The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. CONCLUSIONS This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.

Tissue Engineering in the Trachea

This review summarizes efforts to generate an autologous tissue‐engineered trachea (TET) using various biomaterial or cell sources to make tracheal cartilage to form the structural components of a functional tracheal replacement. Biomechanical assessments of the TET showed that the cartilage stiffness was excellent in the nude models; however, the sheep autologous TET did not provide sufficient support and collapsed easily. As a result, tissue engineering technology is still far from allowing the functional recovery of patients who suffer from severe tracheal disease. On the other hand, there are several clinical reports seeding cells to decellularized tissue using tissue engineering techniques. However, the working mechanisms of the tissue‐engineered trachea remain unclear. Nevertheless, we believe that the field of tissue engineering has great potential for surmounting these obstacles and allowing us to generate functional tracheal replacements in the near future. Anat Rec, 297:44–50. 2014.

Generation of a tissue-engineered tracheal equivalent

In this Minireview we summarize efforts to generate an autologous tissue‐engineered trachea (TET) shaped as a cylinder containing a helix of cartilage to form the structural component of a functional tracheal replacement. Our first step was to demonstrate that a composite engineered tracheal equivalent composed of cartilaginous cylinder could be lined with nasal epithelial cells in a nude‐mouse model. We then demonstrated the feasibility of creating the cartilage and fibrous portion of the trachea using autologous tissue harvested from a single procedure in both nude‐rat and sheep models. Gross morphology and tissue morphology of these TET series were similar to that of native trachea. Histological data indicated the presence of mature cartilage formation of a pseudostratified columnar epithelium and the presence of mature cartilage surrounded by connective tissue, as would be expected of native trachea. Regarding the biomechanical properties, the cartilage was excellent in nude model; however, sheep autologous TET provided less support and therefore collapsed easily. In addition to utilizing the angiogenic qualities of cytokines, we hope to exploit the growth potentials of stromal cells derived from bone marrow for the production of a clinically practical, bioengineered trachea.

Cost-effectiveness associated with the diagnosis and staging of non-small-cell lung cancer.

OBJECTIVE We evaluated how much time and money could be saved without compromising overall results in treating lung cancer. SUBJECTS AND METHODS We retrospectively evaluated 318 patients for T- and M-factors and 335 for N-factor. If bronchoscopy failed to diagnose a mass lesion believed to be malignant in x-ray computed tomography (CT), we proceeded to direct thoracotomy without needle or video-assisted biopsy. When mediastinal nodes were negative in CT, we proceeded to direct thoracotomy without mediastinoscopy. We searched routinely for distant metastasis with brain and abdominal CTs and bone scans. RESULTS Lesions suspected of malignancy in CT were pathologically malignant in 93%. A total of 82.8% of patients with CT-negative mediastinum were without metastasis. The remainder, with metastasis, had a postoperative 5-year survival of 23.5%. Brain CT scans were positive in only 2.2%, abdominal CT scans in 2.4%, and bone scans in 5.0%, for patients with a cT1/T2 non-cN2 lesion. CONCLUSION Brain and abdominal CT scans and bone scans may be omitted for cT1/T2 and non-cN2 lesions in CT. CT-negative mediastinum then leads to direct thoracotomy. The vast majority of patients may thus undergo surgery earlier with less physical and financial burden. The cost saving was calculated to be 59.4% per cT1/T2 non-cN2 patient, or US