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.

Bidirectional developmental potential in reprogrammed cells with acquired pluripotency.

We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP). This reprogramming does not require nuclear transfer or genetic manipulation. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency.

Anesthesia report card - a customizable tool for performance improvement.

PURPOSE: Measuring and providing performance feedback to physicians has gained momentum not only as a way to comply with regulatory requirements, but also as a way to improve patient care. Measurement of structural, process, and outcome metrics in a reliable, evidence-based, specialty-specific manner maximizes the probability of improving physician performance. The manner in which feedback is provided influences whether the measurement tool will be successful in changing behavior. We created an innovative reporting tool template for anesthesiology practitioners designed to provide detailed, continuous feedback covering many aspects of clinical practice. METHODS: The literature regarding quality metric measurement and feedback strategies was examined to design a reporting tool that could provide high quality information and result in improved performance of clinical and academic tasks. A committee of department leaders and information technology professionals was tasked with determining the measurement criteria and infrastructure needed to generate these reports. Data was collected in a systematic, unbiased manner, and reports were populated with information from multiple databases and software systems. Feedback would be based on frequently updated information and allow for analysis of historical performance as well as comparison amongst peers. RESULTS: A template for an anesthesia report card was created. Categories included compliance, credentialing and qualifications, education, clinical and operating room responsibilities, and academic achievements. Physicians were able to choose to be evaluated in some of the categories and had to meet a minimum number of criteria within each category. This allowed for customization to each practitioner's practice. Criteria were derived from the measures of academic and clinical proficiency, as well as quality metrics. Criteria were objective measures and data gathering was often automated. Reports could be generated that were updated daily and provided historical information, and information about peers in the department and within each subspecialty group. CONCLUSIONS: We demonstrate the creation of an online anesthesia report card that incorporates metrics most likely to engender positive changes in practice and academic responsibilities. This tool provides timely and customized information for each anesthesia practitioner, designed to be easily modifiable to improve the quantity, quality, and substance of metrics being measured. Finally, our tool could serve as a template for a performance measuring tool that can be customizable to a wide variety of practice settings, and upon which both monetary and non-monetary incentives might be based in the future.

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.

Adult muscle 'stem' cells can be sustained in culture as free-floating myospheres.

The effectiveness of cell-based therapy to treat muscle disease has been hampered by difficulties in isolating, maintaining and propagating the stem cells that are needed for treatment. Here we report the isolation of muscle-derived stem cells from both young and old mice and their propagation over extended periods of time in culture as "free-floating" myospheres. Analysis of these sphere-forming cells showed that they express stem cell antigen-1 (Sca-1), beta1 integrin (CD29), Thy-1 (CD90), and CD34, but did not express CD45, CD31, or myogenic markers (Pax7, Myf5, and MyoD). We found that cells derived from myospheres and then grown adherently (MDACs) behaved similar to primary myoblasts, in that these cells expressed myogenic markers and were able to easily form multinucleated myotubes. Unlike the parental myospheres but analogous to primary myoblasts, MDACs expressed Pax7, Myf5, and MyoD, indicating that the parent myosphere cells were a more primitive type of cell. In support of this we demonstrated that myospheres were also able to differentiate into adipogenic and osteogenic cells in culture, as well as being able to contribute to injured muscle in vivo. In summary, we report that primitive adult muscle stem cells can be easily isolated and sustained in culture as myospheres.

Tissue-engineered endothelial and epithelial implants differentially and synergistically regulate airway repair.

The trilaminate vascular architecture provides biochemical regulation and mechanical integrity. Yet regulatory control can be regained after injury without recapitulating tertiary structure. Tissue-engineered (TE) endothelium controls repair even when placed in the perivascular space of injured vessels. It remains unclear from vascular repair studies whether endothelial implants recapitulate the vascular epithelial lining or expose injured tissues to endothelial cells (ECs) with unique healing potential because ECs line the vascular epithelium and the vasa vasorum. We examined this issue in a nonvascular tubular system, asking whether airway repair is controlled by bronchial epithelial cells (EPs) or by ECs of the perfusing bronchial vasculature. Localized bronchial denuding injury damaged epithelium, narrowed bronchial lumen, and led to mesenchymal cell hyperplasia, hypervascularity, and inflammatory cell infiltration. Peribronchial TE constructs embedded with EPs or ECs limited airway injury, although optimum repair was obtained when both cells were present in TE matrices. EC and EP expression of PGE(2), TGFbeta1, TGFbeta2, GM-CSF, IL-8, MCP-1, and soluble VCAM-1 and ICAM-1 was altered by matrix embedding, but expression was altered most significantly when both cells were present simultaneously. EPs may provide for functional control of organ injury and fibrous response, and ECs may provide for preservation of tissue perfusion and the epithelium in particular. Together the two cells optimize functional restoration and healing, suggesting that multiple cells of a tissue contribute to the differentiated biochemical function and repair of a tissue, but need not assume a fixed, ordered architectural relationship, as in intact tissues, to achieve these effects.

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.

Lidocaine inhibits NIH-3T3 cell multiplication by increasing the expression of cyclin-dependent kinase inhibitor 1A (p21).

BACKGROUND: We explored molecular mechanisms by which lidocaine inhibits growth in the murine embryonic fibroblast cell line NIH-3T3. Local anesthetics can adversely affect cell growth in vitro. Their effects on wound healing are controversial. We examined the effects and novel mechanisms by which lidocaine affects in vitro multiplication of the murine fibroblast cell line NIH-3T3. METHODS: NIH-3T3 cells were grown in culture with lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM]. Cell multiplication was assessed by determining cell counts on subsequent days, while mechanisms by which inhibition occurred were evaluated by bromodeoxyuridine uptake, gene expression using polymerase chain reaction array, and Western blot analysis to verify increased levels of affected proteins. RESULTS: Lidocaine caused dose-dependent inhibition of multiplication of NIH-3T3 cells. Effects ranged from no inhibition [0.05 and 0.5 mM] and mild inhibition [1 mM], to severe inhibition [2 and 5 mM] [P = 0.006]. Lidocaine 2 mM inhibited bromodeoxyuridine uptake at day 3.5 [P = 0.02 versus control, and P = 0.0495 vs 1 mM lidocaine]. On day 1.5, lidocaine upregulated expression of cyclin-D1 and cyclin-dependent kinase inhibitor 1A [p21]. On day 2.5, lidocaine increased the levels of p21 protein. CONCLUSIONS: Low concentrations of lidocaine, as would be seen in plasma after spinal, epidural, or plexus anesthesia, do not significantly affect multiplication of fibroblasts. Higher doses of lidocaine arrest cell multiplication at the S-phase of the growth cycle by upregulation of p21, an extremely potent inhibitor of cell multiplication. Higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing.

Nanofabrication and microfabrication of functional materials for tissue engineering.

The burgeoning field of regenerative medicine promises significant progress in the treatment of cardiac ischemia, liver disease, and spinal cord injury. Key to its success will be the ability to engineer tissue safely and reliably. Tissue functionality must be recapitulated in the laboratory and then integrated into surrounding tissue upon transfer to the patient. Scaffolding materials must be chosen such that the microenvironment surrounding the cells is a close analog of the native environment. In the early days of tissue engineering, these materials were largely borrowed from other fields, with much of the focus on biocompatibility and biodegradation. However, attention has shifted recently to cell-cell and cell-surface interactions, largely because of enabling technologies at the nanoscale and microscale. Studies on cellular behavior in response to various stimuli are now easily realized by using microfabrication techniques and devices (e.g., biomedical microelectromechanical systems). These experiments are reproducible and moderate in cost, and often can be accomplished at high throughput, providing the fundamental knowledge required to design biomaterials that closely mimic the biological system. It is our opinion that these novel materials and technologies will bring engineered tissues one step closer to practical application in the clinic. This review discusses their application to cardiac, liver, and nerve tissue engineering.

Tissue engineered cartilage "bioshell" protective layer for subcutaneous implants.

OBJECTIVE: One current technique to reconstruct an ear for microtia involves the use of a high density polyethylene auricular implant; however, the implant can extrude if not covered in a temporoparietal fascia flap. Theoretically, an autologous tissue engineered cartilage "bioshell" protective coating around a permanent biocompatible implant might reduce potential extrusion to avoid the flap requirement. We hypothesized that if subjected to intentional exposure, a bioshell coating over an implant would provide enhanced wound healing. METHODS: Six sheets of high density polyethylene and six sheets of 24 carat pure gold wire-mesh measuring 19 mm x 25 mm were implanted subcutaneously in an immunocompetent swine model. Half of each implant group were coated with chondrocytes (50-70 million cells/cm(3)) which were suspended in Pluronic F-127 30% hydrogel; the remaining implants without chondrocytes were used as controls. At 10 weeks post-implantation, partial implant exposure via excision of overlying skin was performed to simulate extrusion and the sites were allowed to heal secondarily. RESULTS: All (6/6) of bioshell implants achieved wound closure after exposure by the seventh post-operative day; controls achieved closure at approximately 10 days. Bioshell neocartilage was evaluated and confirmed histologically using hematoxylin and eosin and safranin O stains. Histochemically, neocartilage approximated native cartilage with 60% glycosaminoglycans content. CONCLUSION: A 'proof-of-principle' tissue engineered bioshell around subcutaneous high density polyethylene and gold implants generated an elastic neocartilage coating, elicited a low inflammatory reaction, and was associated with 30% faster wound healing.

Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs.

Composite tissue-engineered intervertebral tissue was assembled in the shape of cylindrical disks composed of an outer shell of PGA mesh seeded with annulus fibrosus cells with an inner core of nucleus pulposus cells seeded into an alginate gel. Samples were implanted subcutaneously in athymic mice and retrieved at time points up to 16 weeks. At all retrieval times, samples maintained shape and contained regions of distinct tissue formation. Histology revealed progressive tissue formation with distinct morphological differences in tissue formation in regions seeded with annulus fibrosus and nucleus pulposus cells. Biochemical analysis indicated that DNA, proteoglycan, and collagen content in tissue-engineered discs increased with time, reaching >50% of the levels of native tissue by 16 weeks. The exception to this was the collagen content of the nucleus pulposus portion of the implants with were approximately 15% of native values. The equilibrium modulus of tissue-engineered discs was 49.0+/-13.2 kPa at 16 weeks, which was between the measured values for the modulus of annulus fibrosus and nucleus pulposus. The hydraulic permeability of tissue-engineered discs was 5.1+/-1.7x10(-14) m2/Pa at 16 weeks, which was between the measured values for the hydraulic permeability of annulus fibrosus and nucleus pulposus. These studies document the feasibility of creating composite tissue-engineered intevertebral disc implants with similar composition and mechanical properties to native tissue.

Polyglycolic acid-induced inflammation: role of hydrolysis and resulting complement activation.

Tissue and organ replacement have quickly outpaced available supply. Tissue bioengineering holds the promise for additional tissue availability. Various scaffolds are currently used, whereas polyglycolic acid (PGA), which is currently used in absorbable sutures and orthopedic pins, provides an excellent support for tissue development. Unfortunately, PGA can induce a local inflammatory response following implantation. Therefore, we investigated the molecular mechanism of inflammation in vitro and in vivo. Degraded PGA induced an acute peritonitis, characterized by neutrophil (PMN) infiltration following intraperitoneal injection in mice. Similar observations were observed using the metabolite of PGA, glycolide. Dissolved PGA or glycolide, but not native PGA, activated the classical complement pathway in human sera, as determined by classical complement pathway hemolytic assays, C3a and C5a production, and C3 and immunoglobulin deposition. To investigate whether these in vitro observations translated to in vivo findings, we used genetically engineered mice. Intraperitoneal administration of glycolide or dissolved PGA in mice deficient in C1q, factor D, C1q and factor D, or C2 and factor B demonstrated significantly reduced PMN infiltration compared to congenic controls (WT). Mice deficient in C6 also demonstrated acute peritonitis. However, treatment of WT or C6 deficient mice with a monoclonal antibody against C5 prevented the inflammatory response. These data suggest that the hydrolysis of PGA to glycolide activates the classical complement pathway. Furthermore, complement is amplified via the alternative pathway and inflammation is induced by C5a generation. Inhibition of C5a may provide a potential therapeutic approach to limit the inflammation associated with PGA-derived materials following implantation.

A novel approach to regenerating periodontal tissue by grafting autologous cultured periosteum.

In the field of oral and maxillofacial surgery, tissue-engineering techniques have been found useful in regenerating lost tissues. Periodontal disease causes severe destruction of periodontal tissue, including the alveolar bone. In this study we attempted to regenerate canine periodontal tissue defects by grafting autologous cultured membrane derived from the periosteum. Under appropriate culture conditions, periosteal cells produce enough extracellular matrix to form sheets. Periosteum specimens were peeled from the mandibular body of adult hybrid dogs and were cultured until cells formed membrane. ALP activity was measured to determine an optimal time for grafting. The cultured periosteum (CP) was grafted and sutured on a mechanically made Class III furcation defect in the 4th mandibular premolars. After 3 months, the samples were harvested and observed radiologically and histologically. In cases of CP, the bone defects were regenerated and filled with newly formed hard tissue, whereas in the controls the defects remained. These results show that our novel treatment is effective in regenerating alveolar bone for the treatment of periodontal disease.

In vitro culture of chondrocytes in a novel thermoreversible gelation polymer scaffold containing growth factors.

In this study we examined the potential of a novel thermoreversible gelation polymer (TGP) to act as a 3-D hydrogel scaffold and deliver both chondrocytes and growth factors. Chondrocytes obtained from bovine articular cartilage were studied as a suspension in TGP chilled to 4 degrees C, in the presence or absence of the growth factors IGF-1 and/or TGF beta2. The cold cell/aqueous suspensions were injected into a cylindrical mold and cultured at 37 degrees C for up to 16 weeks. Specimens obtained at 12 and 16 weeks were semitranslucent and elastic. The matrices surrounding the chondrocytes were histologically positive to Safranin-O staining and type II collagen staining. The glycosaminoglycan and hydroxyproline contents in the specimens increased as a function of time and because of the presence of growth factors; those cultured with growth factors produced significantly more of these substances than those cultured without. We have concluded that TGP has potential as a scaffold material in the generation of tissue-engineered cartilage in vitro.

Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.

In this study, we describe the isolation and characterization of a population of adult-derived or somatic lung progenitor cells (SLPC) from adult mammalian lung tissue and the promotion of alveolar tissue growth by these cells (both in vitro and in vivo) after seeding onto synthetic polymer scaffolds. After extended in vitro culture, differentiating cells expressed Clara cell 10kDa protein, surfactant protein-C, and cytokeratin but did not form organized structures. When cells were combined with synthetic scaffolds, polyglycolic acid (PGA) or Pluronic F-127 (PF-127), and maintained in vitro or implanted in vivo, they expressed lung-specific markers for Clara cells, pneumocytes, and respiratory epithelium and organized into identifiable pulmonary structures (including those similar to alveoli and terminal bronchi), with evidence of smooth muscle development. Although PGA has been shown to be an excellent polymer for culture of specific cell types in vitro, in vivo culture in an immunocompetent host induced a foreign body response that altered the integrity of the developing lung tissue. Use of PF-127/cell constructs resulted in the development of tissue with less inflammatory reaction. These data suggest that the therapeutic use of engineered tissues requires both the use of specific cell phenotypes, as well as the careful selection of synthetic polymers, to facilitate the assembly of functional tissue.

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.

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 Ham's 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 pseudo-stratified 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.

Tissue engineering: The first decade and beyond.

This article reviews the important developments in the field of tissue engineering over the last 10 years. Research in the area of biomaterials is examined from the perspective of providing the foundation for the development of tissue engineering. Early efforts combining cells with biocompatible materials are described and applications of this technology presented, with particular focus on uses in orthopaedics and maxillofacial surgery. The basic principles of tissue engineering and state-of-the-art technology in cell biology and materials science as used currently in the field are presented. Finally, futures challenges are outlined from the perspective of integrating technologies from medicine, biology, and engineering, in hopes of translating tissue engineering to clinical applications. J. Cell. Biochem. Suppls. 30/31:297-303, 1998. © 1998 Wiley-Liss, Inc.