Nanog-Independent Reprogramming to iPSCs with Canonical Factors

Summary It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem cells and induced pluripotent stem cells (iPSCs). However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever-increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical-reprogramming factors Oct4, Sox2, Klf4, and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog−/− fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPSCs from Nanog−/− fibroblasts that effectively contributed to the germline of chimeric mice. Thus, whereas Nanog may be an important mediator of reprogramming, it is not required for establishing pluripotency in the mouse, even under standard conditions

Developmental bias in cleavage-stage mouse blastomeres

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues

Clinical applications and limitations of current ovarian stem cell research: a review

The publication of a report in Nature in 2004 by the Tilly group suggesting that mouse ovaries are capable of generating oocytes de novo post-natally, has sparked interest in a problem long thought to have been resolved from classical studies in a variety of mammalian species. Within a nearly two year time period, laboratories around the world have taken up the challenge to dogma raised by this initial report, either to test this concept in an experimental basic science setting or give direction to clinical applications that could result, were the original premises of this work in the mouse valid for extrapolation to humans. This review provides a status report for this promising area of research, (1) to summarize recent findings in the literature with respect to the validity of the original hypothesis proffered by the Tilly group, and, (2) to gauge the potential utility of ovarian stem cells as a treatment for certain forms of human infertility

Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia.

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development

Notch Inhibition Allows Oncogene-Independent Generation of iPS Cells

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes ​KLF4 and ​CMYC or the genetic inhibition of the tumor suppressor gene ​p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing ​p21 in a ​p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without ​KLF4 and ​CMYC while leaving ​p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.Molecular and Cellular BiologyStem Cell and Regenerative Biolog

Conversion of Mouse and Human Fibroblasts into Functional Spinal Motor Neurons

The mammalian nervous system is composed of a multitude of distinct neuronal subtypes, each with its own phenotype and differential sensitivity to degenerative disease. Although specific neuronal types can be isolated from rodent embryos or engineered from stem cells for translational studies, transcription factor mediated reprogramming might provide a more direct route to their generation. Here we report that the forced expression of select transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). iMNs displayed a morphology, gene expression signature, electrophysiology, synaptic functionality, in vivo engraftment capacity and sensitivity to degenerative stimuli, similar to embryo-derived motor neurons. We show that the converting fibroblasts do not transit through a proliferative neural progenitor state, and thus form bona fide motor neurons via a route distinct from embryonic development. Our findings demonstrate that fibroblasts can be converted directly into a specific differentiated and functional neural subtype, the spinal motor neuron.Stem Cell and Regenerative Biolog

Reprogramming of Embryonic Human Fibroblasts into Fetal Hematopoietic Progenitors by Fusion with Human Fetal Liver CD34+ Cells

Experiments with somatic cell nuclear transfer, inter-cellular hybrid formation_ENREF_3, and ectopic expression of transcription factors have clearly demonstrated that cell fate can be dramatically altered by changing the epigenetic state of cell nuclei. Here we demonstrate, using chemical fusion, direct reprogramming of the genome of human embryonic fibroblasts (HEF) into the state of human fetal liver hFL CD34+ (hFL) hematopoietic progenitors capable of proliferating and differentiating into multiple hematopoietic lineages. We show that hybrid cells retain their ploidy and can differentiate into several hematopoietic lineages. Hybrid cells follow transcription program of differentiating hFL cells as shown by genome-wide transcription profiling. Using whole-genome single nucleotide polymorphism (SNP) profiling of both donor genomes we demonstrate reprogramming of HEF genome into the state of hFL hematopoietic progenitors. Our results prove that it is possible to convert the fetal somatic cell genome into the state of fetal hematopoietic progenitors by fusion. This suggests a possibility of direct reprogramming of human somatic cells into tissue specific progenitors/stem cells without going all the way back to the embryonic state. Direct reprogramming of terminally differentiated cells into the tissue specific progenitors will likely prove useful for the development of novel cell therapies

Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

Delta-9-tetrahydrocannabinol (THC), the main psychoactive compound of marijuana, induces numerous undesirable effects, including memory impairments, anxiety, and dependence. Conversely, THC also has potentially therapeutic effects, including analgesia, muscle relaxation, and neuroprotection. However, the mechanisms that dissociate these responses are still not known. Using mice lacking the serotonin receptor 5-HT2A, we revealed that the analgesic and amnesic effects of THC are independent of each other: while amnesia induced by THC disappears in the mutant mice, THC can still promote analgesia in these animals. In subsequent molecular studies, we showed that in specific brain regions involved in memory formation, the receptors for THC and the 5-HT2A receptors work together by physically interacting with each other. Experimentally interfering with this interaction prevented the memory deficits induced by THC, but not its analgesic properties. Our results highlight a novel mechanism by which the beneficial analgesic properties of THC can be dissociated from its cognitive side effects

The mouse C9ORF72 ortholog is enriched in neurons known to degenerate in ALS and FTD.

Using transgenic mice harboring a targeted LacZ insertion, we studied the expression pattern of the C9ORF72 mouse ortholog (3110043O21Rik). Unlike most genes that are mutated in amyotrophic lateral sclerosis (ALS), which are ubiquitously expressed, the C9ORF72 ortholog was most highly transcribed in the neuronal populations that are sensitive to degeneration in ALS and frontotemporal dementia. Thus, our results provide a potential explanation for the cell type specificity of neuronal degeneration caused by C9ORF72 mutations

DNA methylation dynamics of the human preimplantation embryo

In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell type-specific regulation directed by DNA binding factors1-3. This comparatively static landscape dramatically contrasts the events of fertilization, where the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, though methylation is maintained at several notable features4-7. While these dynamics have been extensively characterized in the mouse, only limited observations are available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved8-10. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell (ESC) derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of persistent maintenance are primarily restricted to gene bodies. While most features share similar dynamics to mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island (CGI) promoters that extend beyond known Imprint Control Regions (ICRs). Retrotransposon regulation is also highly diverse and transitions from maternally to embryonically expressed, species-specific elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation