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 gene1 (APP; termed APPDp), 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 APPDp 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 APPDp 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.

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense, or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, while the rest were newly occurring during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS safety before clinical use.

Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. In this study, we report that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. Our observations establish a critical role of YAP in maintaining stem cell pluripotency.[Keywords: YAP; Hippo; stem cells; TEAD] Supplemental material is available at http://www.genesdev.org.

BackgroundNeural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).Methodology/Principal FindingsWe performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184+/CD271−/CD44−/CD24+ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184−/CD44−/CD15LOW/CD24+ and a population of glia that was CD184+/CD44+ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.Conclusions/SignificanceThese findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

SUMMARY Predisposition to sporadic Alzheimer’s disease (SAD) involves interactions between a person’s unique combination of genetic variants and the environment. The molecular effect of these variants may be subtle and difficult to analyze with standard in vitro or in vivo models. Here we used hIPSCs to examine genetic variation in the SORL1 gene and possible contributions to SAD-related phenotypes in human neurons. We found that human neurons carrying SORL1 variants associated with an increased SAD risk show a reduced response to treatment with BDNF, at the level of both SORL1 expression and APP processing. shRNA knockdown of SORL1 demonstrates that the differences in BDNF-induced APP processing between genotypes are dependent on SORL1 expression. We propose that the variation in SORL1 expression induction by BDNF is modulated by common genetic variants and can explain how genetic variation in this one locus can contribute to an individual’s risk of developing SAD.

A major goal of stem-cell research is to identify conditions that reliably regulate their differentiation into specific cell types. This goal is particularly important for human stem cells if they are to be used for in vivo transplantation or as a platform for drug development. Here we describe the establishment of procedures to direct the differentiation of human embryonic stem cells and human induced pluripotent stem cells into forebrain neurons that are capable of forming synaptic connections. In addition, HEK293T cells expressing Neuroligin (NLGN) 3 and NLGN4, but not those containing autism-associated mutations, are able to induce presynaptic differentiation in human induced pluripotent stem cellderived neurons. We show that a mutant NLGN4 containing an inframe deletion is unable to localize correctly to the cell surface when overexpressed and fails to enhance synapse formation in human induced pluripotent stem cell-derived neurons. These findings establish human pluripotent stem cell-derived neurons as a viable model for the study of synaptic differentiation and function under normal and disorder-associated conditions. neural differentiation | autism spectrum disorders | synaptogenesis P revious reports have described the differentiation of human ES (hES) cells into neurons (1-6), but they have not directly explored the kinds of in vitro experiments that may be carried out using such neurons. In addition, whether human ES and induced pluripotent stem (hiPS) cell-derived neurons follow the same differentiation programs has not been extensively examined. We sought to devise a method to direct the differentiation of hES and hiPS cells to anterior forebrain fates, as many neurodevelopmental and cognitive disorders, such as autism and schizophrenia, affect forebrain function. Current methodologies are often adapted from mouse ES cell cultures and range from cocultures with a stromal cell line (7,8) to application of recombinant factors and small molecules (3)(4)(5)(9)(10)(11)(12). We show in the present study that we were able to derive forebrain neural progenitor cells (NPCs) from both hES and hiPS cell lines, and that these NPCs can subsequently differentiate into electrophysiologically functional neurons. Furthermore, we applied such hiPS cell-derived forebrain neurons in a bioassay to test the synapse-inducing ability of Neuroligins. Neuroligins comprise a small family of transmembrane synaptic-cell adhesion molecules that are known for their ability to induce artificial synapses in heterologous nonneuronal cells and to modulate synapse numbers when overexpressed in rodent neurons (13). We used this artificial synapse formation assay to compare the synaptogenic potential of two X-linked Neuroligin mutants that have been identified in rare autistic families. In sum, human neurons differentiated in vitro from human pluripotent stem cells may serve as a useful culture system to study the functions of synaptic molecules implicated in neurodevelopmental disorders.

The significance of a population in mouse bone marrow of lineage-negative (Lin−), Sca1-positive, c-kit-negative (LSK−) cells, which is reported to be devoid of long-term repopulation capacity or myeloid potential, is unknown. In this study, we show that the LSK− population is composed of several subsets defined by the expression of flt3, CD25, and IL-7Rα. The first subset was CD25− and more than 90% expressed either flt3, IL-7Rα, or both. The CD25−LSK− population had T cell, B cell, and NK cell potential in vivo, and most of this activity was localized in the flt3+ subset, irrespective of the expression of IL-7Rα. Although lymphoid potential of flt3+LSK− cells in vivo was 3-fold lower than that of lin−Sca1lowkitlowIL7Rα+ common lymphoid progenitors (CLPs), their cloning efficiency in vitro was 10-fold lower than that of CLPs. Furthermore, although the myeloid potential of flt3+LSK− cells was 10-fold lower than that of CLPs in the absence of M-CSF, the relative myeloid potential of both populations was similar in its presence. These observations suggest differential growth factor requirements of both populations. The second subset of LSK− cells was homogeneously CD25+flt3−IL7Rα+ and could be generated from both CD25−LSK− cells and from CLPs, but did not engraft in immunodeficient Rag1−/− or Rag1−/−γc−/− hosts. This population, of which the significance is unclear, was increased in Rag1−/− mice and in old mice. Thus, the LSK− population is phenotypically and functionally heterogeneous and contains early lymphoid-committed precursors. Our findings imply that the early stages of lymphoid commitment are more complex than was thus far assumed.

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