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.

Role for interleukin 1alpha in the inhibition of chondrogenesis in autologous implants using polyglycolic acid-polylactic acid scaffolds.

Significant challenges remain in generating tissue-engineered cartilage in immunocompetent animals. Scaffold materials such as polyglycolic acid lead to significant inflammatory reactions, inhibiting homogeneous matrix synthesis. This study examined the generation of tissue-engineered cartilage, using a polyglycolic acid-polylactic acid copolymer (Ethisorb; Ethicon, Norderstedt, Germany) in an autologous immunocompetent pig model. The goals of this study were to determine the role of interleukin 1alpha (IL-1alpha) in this system and to assess the effect of serum treatment on tissue generation. Porcine auricular chondrocytes were seeded onto Ethisorb disks cultured for 1 week in medium supplemented with either fetal bovine serum or serum-free insulin-transferrin-selenium supplement. Specimens were implanted autogenously in pigs with unseeded scaffolds as controls. After 1, 4, or 8 weeks, six specimens from each group were explanted and analyzed histologically (hematoxylin and eosin, safranin O, trichrome, and Verhoeff's staining) and biochemically (glycosaminoglycan content). The presence and distribution of IL-1alpha were assessed by immunohistochemistry. Histology revealed acute inflammation surrounding degrading scaffold. Cartilage formation was observed as early as 1 week after implantation and continued to increase with time; however, homogeneous matrix synthesis was not present in any of the specimens. Strong IL-1alpha expression was detected in chondrocytes at the implant periphery and in cells in the vicinity of degrading polymer. Histologically there was no significant difference between the experimental groups with respect to the amount of matrix synthesis or inflammatory infiltration. The glycosaminoglycan content was significantly higher in the serum-free group. These results suggest that inflammatory reactions against scaffold materials and serum components lead to the production of cytokines such as IL-1alpha that may inhibit cartilage tissue formation in autologous transplant models.

Microtia chondrocytes as a donor source for tissue-engineered cartilage.

OBJECTIVES/HYPOTHESIS: Current surgical techniques for the correction of microtia are challenging. Research in the field of tissue engineering is providing insight into chondrocyte behavior for a possible future treatment of microtia. The authors wished to evaluate the biological potential of chondrocytes isolated from microtia cartilage as compared with normal auricular cartilage as a source of tissue-engineered cartilage. STUDY DESIGN: A comparative research design to study the potential of microtia cartilage chondrocytes with normal auricular chondrocytes as a source of tissue-engineered cartilage. METHODS: Cartilage specimens from 12 pediatric patients (six normal auricular specimens and six auricular specimens with microtia) were obtained. The chondrocytes were isolated and cultured in vitro; chondrocyte number was increased by passaging. Each type of cell was implanted in nude mice to generate tissue-engineered cartilage. Eight weeks after implantation the specimens were dissected and removed. Results were compared between the normal auricular and microtia specimens in regard to cell number expansion in vitro and generation of tissue-engineered cartilage in vivo. RESULTS: An initial mean cell number of 150,000 cells in each group (normal and microtia) increased to an average cell number of 120 million cells/mL in the normal and 130 million cells in the microtia subgroups, respectively, at the end of the second passage. Histologically, both types of chondrocytes generated normal elastic cartilage. CONCLUSION: The study demonstrated the potential of cells isolated from microtia cartilage to generate tissue-engineered cartilage. Microtia cartilage represents an important additional donor source for the possible generation of a human tissue-engineered auricle.

Tissue-engineered cartilage as a graft source for laryngotracheal reconstruction: a pig model.

OBJECTIVE: To evaluate the feasibility of using tissue-engineered cartilage for laryngotracheal reconstruction in the pig model. DESIGN: Auricular cartilage was harvested from 3 young swine. The cartilage was digested, processed, and suspended and a cell culture was obtained. The cells were then suspended in 3 mL of a 30% solution of a biodegradable polymer (Pluronic F-127) (polyethylene oxide/polypropylene oxide copolymer) at a cellular concentration of 50 x 10(6) cells/mL. This suspension was then implanted subcutaneously into each pig's dorsum. Eight weeks after implantation, the cartilage was harvested with the surrounding perichondrial capsule. An anterior cartilage graft laryngotracheal reconstruction was performed. Bronchoscopy was performed at 3 postoperative weeks to demonstrate airway patency. The animals were killed at 3 months, and specimens were obtained for histological analysis. SETTING: An animal research facility. SUBJECTS: Three young Yorkshire swine. RESULTS: All 3 pigs survived to the 3-month postoperative interval with no evidence of stridor or airway distress. Interval bronchoscopy revealed a normal patent airway with a mucosalized graft. Histopathologic analysis revealed incorporation of the tissue-engineered cartilage graft in the cricoid area, which correlated with results of bronchoscopic evaluation. CONCLUSION: Tissue-engineered auricular cartilage served as a viable graft in the pig model and might be an alternative cartilage source for laryngotracheal reconstruction.

Novel technique for peripheral nerve reconstruction in the absence of an artificial conduit.

The purpose of this study is to promote nerve regeneration across a peripheral nerve gap, using a biologic, tissue-engineered nerve (TEN), containing a high density of viable Schwann cells (SCs) in the absence of supportive foreign materials and a tubular system. Isolated SCs from adult rat sciatic nerve were seeded onto biodegradable constructs and implanted into the backs of nude mice to create TENs. Six weeks later, the constructs were harvested, implanted into surgically created sciatic nerve gaps in rats without supportive artificial conduits and compared with both an autograft group and a silicone conduit group using SCs. Two months later, functional assessment was evaluated by walking track analysis and the implanted lesions were imaged by transmission electron microscopy. The axonal number and sciatic function index of the TEN were significantly higher than those of the silicone group and achieved a comparable level to the autograft group. The results indicate that the large number of SCs within their own extracellular matrix appeared sufficient to enable neuronal growth across a nerve gap in the absence of an artificial conduit and that these circumstances may have a positive effect on the supplement of growth factors from the surrounding tissues of implanted TEN.

Normal features of tissue-engineered auricular cartilage by flow cytometry and histology: patient safety.

BACKGROUND: Cytokinetic abnormalities in DNA content, such as aneuploidy, haploidy, and tetraploidy, have been found to occur in human cartilaginous tumors. The high number of chondrocytes needed for tissue-engineered cartilaginous implants requires the cells to be passaged repeatedly. The theoretical risk of changes in the normal diploid state of these cells during their growth in vitro and after generation of tissue-engineered cartilage in vivo is not known. Materials and methods Auricular chondrocytes were obtained from 6 patients and cultured in vitro. Chondrocyte number was increased by repeated passaging. The passaged cells were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Fresh control chondrocytes along with the passaged cells and cells obtained from the tissue-engineered constructs were collected and compared for DNA content by flow cytometry. RESULTS: Flow cytometry demonstrated 100% diploidy with no evidence of aneuploidy, haploidy, or tetraploidy in all groups of cells. Histology of the tissue-engineered cartilage also showed no evidence of cellular atypia. CONCLUSION: The number of human auricular chondrocytes can be increased by repeated passaging and passaged chondrocytes can be safely used for implantation to generate tissue-engineered constructs without a change in the normal diploid state of the cells. Histology of the cartilage generated showed normal features without atypia.

A novel technique to isolate adult Schwann cells for an artificial nerve conduit.

The use of an artificial nerve conduit containing viable Schwann cells (SCs) is one of the most promising approaches to repair nerve injuries. Obtaining a large number of viable SCs in a short period is demanded for the clinical use of this technique. However, the previous methods using mitogens are not clinically acceptable, and other methods that do not require mitogens, failed to isolate adult SCs effectively or required a long period of time. In this study, we have developed a novel technique to isolate SCs from adult rat peripheral nerves for an artificial nerve conduit without mitogens, which has produced a total number of 1.21 x 10(5) cells per mg, with an average purity of 93.0+/-0.58% at 21 days in vitro. The Bottenstein-Sato (BS) medium used in this study, had originally been developed for oligodendrocyte culture, but here it is shown to have an effect on SC proliferation and survival. By changing fetal bovine serum (FBS) concentrations from 0 to 10% serially, SCs could be isolated maximally from the predegenerated nerves while suppressing fibroblast overgrowth. The combination of this technique and the altered medium promoted the migration and proliferation of SCs selectively by utilizing the supporting cells of SCs instead of discarding them by changing the culture dishes and media.

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. METHODS: Scaffolds were created in the shape of five full-sized human auricles and five nasal tip cartilaginous skeletons. Bovine shoulder chondrocytes at a concentration of 50 million cells/mL were seeded onto the scaffolds and were grown for 12 weeks in vitro. Two of the auricular scaffolds had internal support provided by soft acrylic sheets and were later implanted into nude rats. RESULTS: All of the scaffolds maintained shape and size through 12 weeks of in vitro culture. On gross examination the scaffolds were progressively replaced by cartilage, which was confirmed by histological and biochemical analysis. The auricular scaffolds with the acrylic internal support had the most natural rigidity, which was observed by gentle palpation. The nasal scaffolds maintained excellent definition even without internal support. CONCLUSION: An adult human-sized auricle and nasal tip cartilaginous structure can be grown entirely in vitro using principles of tissue engineering.