No abstract

A common obstacle to the survival of encapsulated tissue is oxygen insufficiency. This appears particularly true of encapsulated pancreatic β-cells. Our work investigates a fluorescent hypoxia detection system for early recognition of hypoxic stress in encapsulated pancreatic tissue. Murine insulinoma (MIN6) cells were engineered to produce a red fluorescent protein under the control of hypoxia-inducible-factor-1. Aggregates of these cells were encapsulated in poly(ethylene glycol) hydrogels at densities of 200,000, 600,000, and 1 million cells per capsule then incubated in either a 1% or 20% oxygen environment. Cell function was evaluated by daily measurement of glucose-stimulated insulin secretion. Encapsulated cells were also fluorescently imaged periodically over 72 h for expression of the marker signal. Results indicate that oxygen insufficiency severely impacts insulin release from MIN6 cells, and that large aggregates are especially vulnerable to oxygen limitations. Our marker was found to be successfully indicative of hypoxia and could be used as a predictor of subsequent insulin release. Further work will be required to fully characterize signal dynamics and to evaluate in vivo efficacy. The method presented here represents a unique and valuable approach to detecting hypoxic stress in living tissues which may prove useful to a variety of fields of biological research.

Tissue engineering is a promising approach to repair critical-size defects in bone. Damage to vasculature at the defect site can create a lower O2 environment compared with healthy bone. Local O2 levels influence stem cell behavior, as O2 is not only a nutrient, but also a signaling molecule. The hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates a wide range of O2-related genes and its contribution in bone repair/formation is an important area that can be exploited. In this study, we examined the effect of low O2 environments (1% and 2% O2) on the osteogenic differentiation of adipose-derived stem cells in both two-dimensional (2-D) and three-dimensional (3-D) culture systems. To determine the role of HIF-1 in the differentiation process, an inhibitor was used to block the HIF-1 activity. The samples were examined for osteogenesis markers as measured by quantification of the alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteonectin (ON) and osteopontin (OPN). Results show a downregulation of the osteogenic markers (ALP activity, mineralization, ON, OPN) in both 1% and 2% O2 when compared to 20% O2 in both 2-D and 3-D culture. Vascular endothelial growth factor secretion over 28 days was significantly higher in low O2 environments and HIF-1 inhibition reduced this effect. The inhibition of the HIF-1 activity did not have a significant impact on the expression of the osteogenic markers, suggesting HIF-1-independent inhibition of osteogenic differentiation in hypoxic conditions.

Oxygen is not only a nutrient but also an important signaling molecule whose concentration can influence the fate of stem cells. This study details the development of a marker of hypoxic signaling for use with encapsulated cells. Testing of the marker was performed with adipose-derived stem cells (ADSCs) in two-dimensional (2D) and 3D culture conditions in varied oxygen environments. The cells were genetically modified with our hypoxia marker, which produces a red fluorescent protein (DsRed-DR), under the control of a hypoxia-responsive element (HRE) trimer. For 3D culture, ADSCs were encapsulated in poly(ethylene glycol)-based hydrogels. The hypoxia marker (termed HRE DsRed-DR) is built on a recombinant adenovirus and ADSCs infected with the marker will display red fluorescence when hypoxic signaling is active. This marker was not designed to measure local oxygen concentration but rather to show how a cell perceives its local oxygen concentration. ADSCs cultured in both 2D and 3D were exposed to 20% or 1% oxygen environments for 96 h. In 2D at 20% O 2 , the marker signal was not observed during the study period. In 1% O 2 , the fluorescent signal was first observed at 24 h, with maximum prevalence observed at 96 h as 59% -3% cells expressed the marker. In 3D, the signal was observed in both 1% and 20% O 2 . The onset of signal in 1% O 2 was observed at 4 h, reaching maximum prevalence at 96 h with 76% -4% cells expressing the marker. Interestingly, hypoxic signal was also observed in 20% O 2 , with 13% -3% cells showing positive marker signal after 96 h. The transcription factor subunit hypoxia inducible factor-1a was tracked in these cells over the same time period by immunostaining and western blot analysis. Immunostaining results in 2D correlated well with our marker at 72 h and 96 h, but 3D results did not correlate well. The western blotting results in 2D and 3D correlated well with the fluorescent marker. The HRE DsRed-DR virus can be used to track the onset of this response for encapsulated, mesenchymal stem cells. Due to the importance of hypoxic signaling in determination of stem cell differentiation, this marker could be a useful tool for the tissue engineering community.

Immobilizing hydrated soft tissue specimens for atomic force microscopy (AFM) is a challenge. Here, we describe a simple and very cost-effective immobilization method, based on the use of transglutaminase in an aqueous environment, and successfully apply it to AFM characterization of human native Wharton's Jelly (nWJ), the gelatinous connective tissue matrix of the umbilical cord. A side-by-side comparison with a widely used polyphenolic protein-based tissue adhesive (Corning Cell-Tak), which is known to bind strongly to virtually all inorganic and organic surfaces in aqueous environments, shows that both adhesives successfully immobilize nWJ in its physological hydrated state. The cost of transglutaminase, however, is over 3000-fold lower than that of Cell-Tak, making it a very attractive method for immobilizing soft tissues for AFM characterization.