Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

Stem Cell Surface Marker Expression Defines Late Stages of Reprogramming to Pluripotency in Human Fibroblasts.

UNLABELLED: Our current understanding of the induction of pluripotency by defined factors indicates that this process occurs in discrete stages characterized by specific alterations in the cellular transcriptome and epigenome. However, the final phase of the reprogramming process is incompletely understood. We sought to generate tools to characterize the transition to a fully reprogramed state. We used combinations of stem cell surface markers to isolate colonies emerging after transfection of human fibroblasts with reprogramming factors and then analyzed their expression of genes associated with pluripotency and early germ lineage specification. We found that expression of a subset of these genes, including the cell-cell adhesion molecule CDH3, characterized a late stage in the reprogramming process. Combined live-cell staining with the antibody GCTM-2 and anti-CDH3 during reprogramming identified colonies of cells that showed gene expression patterns very similar to those of embryonic stem cell or established induced pluripotent stem cell lines, and gave rise to stable induced pluripotent stem cell lines at high frequency. Our findings will facilitate studies of the final stages of reprogramming of human cells to pluripotency and will provide a simple means for prospective identification of fully reprogrammed cells. SIGNIFICANCE: Reprogramming of differentiated cells back to an embryonic pluripotent state has wide ranging applications in understanding and treating human disease. However, how cells traverse the barriers on the journey to pluripotency still is not fully understood. This report describes tools to study the late stages of cellular reprogramming. The findings enable a more precise approach to dissecting the final phases of conversion to pluripotency, a process that is particularly poorly defined. The results of this study also provide a simple new method for the selection of fully reprogrammed cells, which could enhance the efficiency of derivation of cell lines for research and therapy.

State-of-the-art modified RNAi compounds for therapeutics.

In recent years, the recognition of non-protein coding RNAs as a functional effector of genetic expression has been highlighted by the discovery of RNA interference (RNAi). RNAi is an intracellular phenomenon that enables the eukaryotic cell to utilize double-stranded RNA molecules to silence gene expression in a sequence-specific manner. The short interfering RNA (siRNA) pathway has been intensively investigated and continues to serve as the basis for the development of potent molecular genetic tools. The power of this technology is most clearly evidenced by the fact that siRNA effector molecules can be chemically synthesized and exogenously delivered to specifically target and silence any gene of choice. This capability enables not only basic research, but also opens the door to a new therapeutic modality. Furthermore, the introduction of certain chemical modifications to siRNA effectors can produce a more robust knockdown of gene expression, hence, optimizing serum stability and increasing target specificity yet limiting the induction of cellular stress response, which are key features for in vivo delivery and successful therapeutics. This article outlines the progress in the development of differentially modified siRNA duplexes and their potential role as human therapeutics.

Induction of the acrosome reaction and zona-free hamster oocyte penetration by a bull with complete teratospermia versus a half brother with normal sperm.

A fertile bull producing normal sperm and a sterile half brother exhibiting 100% teratospermia were available to study an induced sperm acrosome reaction and oocyte penetration. Pedigree analysis indicated that this condition was inherited. Experiments were undertaken to study the induction of the acrosome reaction using dilaurylphosphatidylcholine (PC12) liposomes, because this procedure was previously established to be highly correlated with bull fertility. The sperm from each bull were incubated with several PC12 concentrations for varying time periods. The initial percentages of sperm from the sterile bull with intact, partially intact, and lost acrosomes were 67%, 18%, and 14%, respectively, vs 82%, 13%, and 5% for the fertile bull (P < .05). After incubation for 15 minutes with 50 microM PC12 liposomes the corresponding values were, respectively, 51%, 26%, and 19%; and 60%, 28%, and 12%. Thus, the differences after induction of the acrosome reaction, although significant (P < .05), were small. The number of sperm adhered to each oocyte averaged 22 and 10, respectively, for the fertile and sterile bulls, whereas 74% of the fertile bull sperm and only 11% of the sterile bull sperm penetrated oocytes. Mixing the sperm-oocyte complex during incubation and increasing the sperm concentration during incubation to compensate for differences in sperm motility did not markedly affect oocyte penetration by teratogenic sperm, which is consistent with this bull being sterile. In other studies, microinjection of this type of sperm was demonstrated to induce fertilization, so the consequences of using sperm with hereditary defects in assisted reproductive programs to overcome human male sterility may be a concern.

Single-cell gene expression profiles define self-renewing, pluripotent, and lineage primed states of human pluripotent stem cells.

Pluripotent stem cells display significant heterogeneity in gene expression, but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states, even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity, consistently high transcripts for all pluripotency-related genes studied, and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states, which ultimately become primed for lineage specification.

Functional characterization of Friedreich ataxia iPS-derived neuronal progenitors and their integration in the adult brain.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.

A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells.

BACKGROUND: Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment. METHODOLOGY/PRINCIPAL FINDINGS: We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3. SIGNIFICANCE: These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes) characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency.

Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog.

RNA interference (RNAi) holds great promise as a tool to study the basic biology of stem cells or to direct differentiation in a specific manner. Barriers to achieving efficient and specific gene silencing in RNAi experiments include limitations in transfection efficiency and in the efficacy and specificity of RNAi silencing effectors. Here, we combine methods of efficient lipid-mediated delivery with chemically modified RNAi compounds to silence genes related to pluripotency, in order to direct differentiation of mouse embryonic stem cells. After transfection of embryonic stem cells with OCT4- or Nanog-targeted RNAi compounds, levels of OCT4 or Nanog transcript and protein were reduced accordingly. Reduction in OCT4 expression correlated with induction of trophectoderm genes Cdx2, Hand1, and PL-1, with formation of cells with trophoblast giant cell phenotype after 6 days. Reduction in Nanog expression correlated with induction of extraembryonic endoderm genes GATA4, GATA6, and laminin B1, with subsequent generation of groups of cells with parietal endoderm phenotype. Our results indicate that transient inhibition of OCT4 or Nanog by RNAi compounds is sufficient to induce differentiation toward extraembryonic lineages, which supports the model that these transcription factors function in a dose-dependent manner to influence cell fate.