Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations

Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies

DNA Methylation

<p><b>A</b>. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s009" target="_blank">S6A Table</a>) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. <b>B</b>. DNA methylation at imprinted loci. Methylation levels for imprinted probes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s009" target="_blank">S6B Table</a>) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. <b>C, D, E</b>. Heatmaps showing differential DNA methylation genes for early vs. late passage <b>(C)</b>, mechanical vs. enzymatic passage <b>(D)</b>, and Mef vs. Ecm substrate <b>(E)</b>. In heatmap <b>(C)</b>, the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.ref013" target="_blank">13</a>]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).</p