A More Accurate Method to Estimate Aminoglycoside Clearance From Serum Creatinine in Patients With Spinal Cord Injury.

Background: Current literature reports that the traditional methods overestimate renal function in spinal cord injury (SCI); however, there is no accepted standard method. Objective: This study evaluated 6 published methods against measured aminoglycoside (AG) drug clearance and determined the frequency with which each method would achieve target peak and trough AG concentrations within a specified range. Methods: A chart-based investigation was conducted at a hospital with a large SCI population, and a total of 35 patients met the inclusion criteria: a diagnosis of long-standing SCI, administration of AG via intravenous infusion, and at least one set of steady-state AG peak and trough concentrations. Pharmacokinetic analysis was performed to compare the measured AG clearance values against the values resulting from 6 methods of estimating the glomerular filtration rate (GFR). Patient-specific pharmacokinetic parameters were used to simulate steady-state peak and trough AG concentrations from doses derived from each method. Results: Compared with the other methods, the Lee-Dang method was found to be more accurate, with the smallest magnitude of variance from the measured AG clearance values. Five alternative methods significantly overestimated AG clearance, by approximately 70% to 160% (P < .05). The Lee-Dang method underestimated AG clearance (by 10%), however not to a significant degree (P = .079). Compared with the alternative methods, the Lee-Dang method resulted in a higher frequency of steady-state peak and trough AG concentrations within the target range specified. Conclusion: The Lee-Dang equation for predicting GFR was more accurate relative to the other methods in the study population of patients with long-term SCI.

SSEA4 is a potential negative marker for the enrichment of human corneal epithelial stem/progenitor cells.

PURPOSE: To examine the expression of stage-specific embryonic antigen-4 (SSEA4) in the epithelium of the human ocular surface and characterize SSEA4(+) and SSEA4(-) limbal epithelial cells. METHODS: SSEA4 expression in the human cornea and limbus was examined by RT-PCR and immunohistochemistry. SSEA4(+) and SSEA4(-) cells were then separated by using magnetic beads. The phenotypes of these two cell populations were evaluated on the basis of cell size, clonogenic assay, and expression of putative limbal stem cell (LSC) and corneal epithelial differentiation markers. RESULTS: SSEA4 was expressed in all layers of the corneal and anterior limbal epithelia. Discrete clusters of SSEA4(+) cells were present in the central and posterior limbal epithelia. SSEA4(+) cells accounted for an average of 40% of the total limbal epithelial cells. The SSEA4(-) population contained five times more small cells (≤11 µm in diameter) than did the SSEA4(+) population. The expression levels of the putative LSC markers ABCG2, ΔNp63α, and cytokeratin (K)14 were significantly higher in the SSEA4(-) population than in the SSEA4(+) population. The SSEA4(-) cells also expressed a significantly higher level of N-cadherin, but a lower level of the differentiation marker K12. The colony-forming efficiency in the SSEA4(-) population was 25.2% (P = 0.04) and 1.6-fold (P < 0.05) higher than in the unsorted population and the SSEA4(+) population, respectively. CONCLUSIONS: SSEA4 is highly expressed in differentiated corneal epithelial cells, and SSEA4(-) limbal epithelial cells contain a higher proportion of limbal stem/progenitor cells. SSEA4 could be used as a negative marker to enrich the isolation of LSCs.

Wnt/ß-catenin signaling regulates proliferation of human cornea epithelial stem/progenitor cells.

PURPOSE: To investigate the expression and role of the Wnt signaling pathway in human limbal stem cells (LSCs). METHODS: Total RNA was isolated from the human limbus and central cornea. Limbal or cornea-specific transcripts were identified through quantitative real-time PCR. Protein expression of Wnt molecules was confirmed by immunohistochemistry on human ocular tissue. Activation of Wnt signaling using lithium chloride was achieved in vitro and its effects on LSC differentiation and proliferation were evaluated. RESULTS: Expression of Wnt2, Wnt6, Wnt11, Wnt16b, and four Wnt inhibitors were specific to the limbal region, whereas Wnt3, Wnt7a, Wnt7b, and Wnt10a were upregulated in the central cornea. Nuclear localization of ß-catenin was observed in a very small subset of basal epithelial cells only at the limbus. Activation of Wnt/ß-catenin signaling increased the proliferation and colony-forming efficiency of primary human LSCs. The stem cell phenotype was maintained, as shown by higher expression levels of putative corneal epithelial stem cell markers, ATP-binding cassette family G2 and ΔNp63α, and low expression levels of mature cornea epithelial cell marker, cytokeratin 12. CONCLUSIONS: These findings demonstrate for the first time that Wnt signaling is present in the ocular surface epithelium and plays an important role in the regulation of LSC proliferation. Modulation of Wnt signaling could be of clinical application to increase the efficiency of ex vivo expansion of corneal epithelial stem/progenitor cells for transplantation.

Activin A-induced differentiation of embryonic stem cells into endoderm and pancreatic progenitors-the influence of differentiation factors and culture conditions.

The differentiation of murine and human embryonic stem (ES) cells into pancreatic cell types has been shown by several methods including spontaneous differentiation, formation of multi-lineage progenitors, lineage selection or transgene expression. However, these strategies led to a mixture of cells of all three primary germ layers and only a low percentage of definitive endoderm cells giving rise to pancreas, liver, lung and intestine. To reproducibly generate functional insulin-producing cells, ES cells have to be differentiated via definitive endoderm and pancreatic endocrine progenitors recapitulating the in vivo development. Activin A, a member of the transforming growth factor beta superfamily, has been shown to induce definitive endoderm cells dependent on concentration, culture conditions and time of application. Moreover, serum components or contamination by feeder cells as well as differentiation and proliferation factors are critical for successful generation of activin A-induced ES cells into endoderm and pancreatic cells. The review presents an overview on those factors that influence activin A activity on endoderm and endocrine progenitor cells and determines the role of signaling factors in the differentiation process into the pancreatic lineage.

Differentiation analysis of pluripotent mouse embryonic stem (ES) cells in vitro.

Pluripotent embryonic stem (ES) cells are characterized by their almost unlimited potential to self-renew and to differentiate into virtually any cell type of the organism. Here we describe basic protocols for the in vitro differentiation of mouse ES cells into cells of the cardiac, neuronal, pancreatic, and hepatic lineage. The protocols include (1) the formation of embryoid bodies (EBs) followed by (2) the spontaneous differentiation of EBs into progenitor cells of the ecto-, endo-, and mesodermal germ layer and (3) the directed differentiation of early progenitors into the respective lineages. Differentiation induction via growth and extracellular matrix factors leads to titin-expressing spontaneously beating cardiac cells, tyrosine hydroxylase-expressing dopaminergic neurons, insulin and c-peptide co-expressing pancreatic islet-like clusters, and albumin-positive hepatic cells, respectively. The differentiated cells show tissue-specific proteins and electrophysiological properties (action potentials and ion channels) in cardiac and neuronal cells, glucose-dependent insulin release in pancreatic cells, or glycogen storage and albumin synthesis in hepatic cells. The protocols presented here provide basic systems to study differentiation processes in vitro and to establish strategies for the use of stem cells in regenerative therapies.