Kristina Detmer

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-κB expression

Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury. Nuclear factor (NF)-κB is thought to regulate the expression of endotoxin [lipopolysaccharide (LPS)]-induced inflammatory cytokines such as TNF, and NF-κB may also be influenced by changes in O2tension. It is thus proposed that acute changes in O2 tension surrounding AMs alter NF-κB activation and TNF secretion in these lung cells. AM-derived TNF secretion and NF-κB expression were determined after acute hypoxic exposure of isolated Sprague-Dawley rat AMs. Adhered AMs (106/ml) were incubated (37°C at 5% CO2) for 2 h with LPS ( Pseudomonas aeruginosa, 1 μg/ml) in normoxia (21% O2-5% CO2) or hypoxia (1.8% O2-5% CO2). AM-derived TNF activity was measured with a TNF-specific cytotoxicity assay. Electrophoretic mobility shift and supershift assays were used to determine NF-κB activation and to identify NF-κB isoforms in AM extracts. In addition, mRNAs for selected AM proteins were determined with RNase protection assays. LPS-exposed AMs in hypoxia had higher levels of TNF ( P < 0.05) and enhanced expression of NF-κB ( P < 0.05); the predominant isoforms were p65 and c-Rel. Increased mRNA bands for TNF-α, interleukin-1α, and interleukin-1β were also observed in the hypoxic AMs. These results suggest that acute hypoxia in the lung may induce enhanced NF-κB activation in AMs, which may result in increased production and release of inflammatory cytokines such as TNF.

Isoproterenol Inhibits Transcription of Cardiac Cytokine Genes Induced by Reactive Oxygen Intermediates

Background Cytokines such as tumor necrosis factor &agr; (TNF-&agr;) are produced by the myocardium in heart disease and might be stimulated by reactive oxygen. In some cell types, cyclic adenosine monophosphate (AMP) inhibits TNF-&agr; production. The authors tested the hypothesis that stimulation of cardiac &bgr;-adrenergic receptors would inhibit cytokine gene transcription induced by reactive oxygen. Methods Rat hearts were perfused with buffer containing hypoxanthine. Reactive oxygen intermediates were generated by infusion of xanthine oxidase. Myocardial mRNA encoding 11 cytokines was determined. TNF-&agr;, interleukin-6, and cyclic AMP were measured in the coronary effluent. Results In control hearts, of the screened RNA, only mRNA encoding interleukin-1&bgr;, -4, and -6 was detected. Stimulation with hypoxanthine–xanthine oxidase (HX–XO) induced detectable mRNA for TNF-&agr; and interleukin-5 and increased mRNA band density for interleukin-1&bgr;, -4, and -6. Simultaneous infusion of isoproterenol inhibited HX–XO-stimulated cytokine gene expression and caused release of cyclic AMP into the coronary effluent. In control hearts, TNF-&agr; was not detected in the coronary effluent. After HX–XO administration, TNF-&agr; was reliably detected at 60 min and interleukin-6 at 90 min. Simultaneous infusion of isoproterenol inhibited TNF-&agr; and interleukin-6 release. Inclusion of propranolol in the perfusion buffer blocked the isoproterenol-induced inhibition of HX–XO-stimulated TNF-&agr; release and release of cyclic AMP into the coronary effluent. In addition, elevating myocardial cyclic AMP with forskolin also blocked release of TNF-&agr; stimulated by HX–XO. Finally, delaying infusion of isoproterenol until 30 min after HX–XO administration still suppressed release of TNF-&agr;. Conclusions Reactive oxygen species activate cytokine gene transcription in the myocardium. The sympathetic nervous system, acting through &bgr;-receptors to elevate myocardial cyclic AMP, regulates cardiac cytokine production by inhibition of transcription.

Cell Surface Marker and Cell Cycle Analysis, Hedgehog Signaling, and Flow Cytometry

Detailed cytological analysis of cells undergoing differentiation often reveals clues to the regulation of multiple cell features. The Hedgehog (Hh) signaling cascade is a master regulator of cell fate during differentiation and is implicated in the development of some neoplasias. Hh signaling affects the expression of cell surface markers of differentiation. We have used the flow cytometer to evaluate the effect of blockage of the Hh signal on the expression of cell surface markers of erythroid differentiation in an in vitro system. In addition, the effect of Hh signaling on the distribution of cells in the phases of the cell cycle over the course of erythroid differentiation was assessed. Inhibition of the Hh signal retards progression of the erythroid developmental program. Included is a discussion of some of the basic parameters, limitations, and interpretations of flow cytometric analysis used for CD marker expression and cell cycle studies.