Transcriptome Dynamics of Human Neuronal Differentiation From iPSC

The generation and use of induced pluripotent stem cells (iPSCs) in order to obtain all differentiated adult cell morphologies without requiring embryonic stem cells is one of the most important discoveries in molecular biology. Among the uses of iPSCs is the generation of neuron cells and organoids to study the biological cues underlying neuronal and brain development, in addition to neurological diseases. These iPSC-derived neuronal differentiation models allow us to examine the gene regulatory factors involved in such processes. Among these regulatory factors are long non-coding RNAs (lncRNAs), genes that are transcribed from the genome and have key biological functions in establishing phenotypes, but are frequently not included in studies focusing on protein coding genes. Here, we provide a comprehensive analysis and overview of the coding and non-coding transcriptome during multiple stages of the iPSC-derived neuronal differentiation process using RNA-seq. We identify previously unannotated lncRNAs via genome-guided de novo transcriptome assembly, and the distinct characteristics of the transcriptome during each stage, including differentially expressed and stage specific genes. We further identify key genes of the human neuronal differentiation network, representing novel candidates likely to have critical roles in neurogenesis using coexpression network analysis. Our findings provide a valuable resource for future studies on neuronal differentiation.

daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development

Production of proliferative Follicle Cells (FCs) and quiescent Escort Cells (ECs) by Follicle Stem Cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (Cap Cells, ECs) and posterior (polar FCs) sources. Here we show that ECs, FSCs and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs and most FCs derive from Intermingled Cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.

daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Stem Cells Applications in Therapeutics and Site-Specific Genome Editing Through CRISPR Cas9 System

Stem cells are immature cells that have ability to differentiate into all specific and mature cells in body. The two main characteristics of stem cells are self-renewable and ability to differentiate into all mature, functional and adult cells types. There are the two major classes a) pluripotent stem cells which have potential to differentiate in all adult cell and b) multipotent stem cells which have capacity to differentiate into many adult cells but not in all cell types.

A Perspective on Adventitious Root Formation in Tree Species

Adventitious root formation is an organogenic process, regulated at several levels, that is crucial for the successful vegetative propagation of numerous plants. In many tree species, recalcitrance to adventitious root formation is a major limitation in the clonal propagation of elite germplasms. Information on the mechanisms underlying the competence for adventitious root formation is still limited. Therefore, increasing our understanding of the mechanisms that enable differentiated somatic cells to switch their fates and develop into root meristematic cells, especially those involved in cell developmental aging and maturation, is a priority in adventitious root-related research. The dynamic cell wall–cytoskeleton, along with soluble factors, such as cellular signals or transcriptional regulators, may be involved in adult cell responses to intrinsic or extrinsic factors, resulting in maintenance, induction of root meristematic cell formation, or entrance into another differentiating pathway.

Unraveling the adult cell progeny of early postnatal progenitor cells

NG2-glia, also referred to as oligodendrocyte precursor cells or polydendrocytes, represent a large pool of proliferative neural cells in the adult brain that lie outside of the two major adult neurogenic niches. Although their roles are not fully understood, we previously reported significant clonal expansion of adult NG2-cells from embryonic pallial progenitors using the StarTrack lineage-tracing tool. To define the contribution of early postnatal progenitors to the specific NG2-glia lineage, we used NG2-StarTrack. A temporal clonal analysis of single postnatal progenitor cells revealed the production of different glial cell types in distinct areas of the dorsal cortex but not neurons. Moreover, the dispersion and size of the different NG2 derived clonal cell clusters increased with age. Indeed, clonally-related NG2-glia were located throughout the corpus callosum and the deeper layers of the cortex. In summary, our data reveal that postnatally derived NG2-glia are proliferative cells that give rise to NG2-cells and astrocytes but not neurons. These progenitors undergo clonal cell expansion and dispersion throughout the adult dorsal cortex in a manner that was related to aging and cell identity, adding new information about the ontogeny of these cells. Thus, identification of clonally-related cells from specific progenitors is important to reveal the NG2-glia heterogeneity.

YAP–TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP–TEAD respond to cell–cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell–cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP–TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP–TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP–TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.

Reversine: A Synthetic Purine with a Dual Activity as a Cell Dedifferentiating Agent and a Selective Anticancer Drug

The development of new therapeutic applications for adult and embryonic stem cells has dominated regenerative medicine and tissue engineering for several decades. However, since 2006, induced Pluripotent Stem Cells (iPSCs) have taken center stage in the field, as they promised to overcome several limitations of the other stem cell types. Nonetheless, other promising approaches for adult cell reprogramming have been attempted over the years, even before the generation of iPSCs. In particular, two years before the discovery of iPSCs, the possibility of synthesizing libraries of large organic compounds, as well as the development of high-throughput screenings to quickly test their biological activity, enabled the identification of a 2,6-disubstituted purine, named reversine, which was shown to be able to reprogram adult cells to a progenitor-like state. Since its discovery, the effect of reversine has been confirmed on different cell types, and several studies on its mechanism of action have revealed its central role in inhibitory activity on several kinases implicated in cell cycle regulation and cytokinesis. These key features, together with its chemical nature, suggested a possible use of the molecule as an anti-cancer drug. Remarkably, reversine exhibited potent cytotoxic activity against several tumor cell lines in vitro and a significant effect in decreasing tumor progression and metastatization in vivo. Thus, 15 years since its discovery, this review aims at critically summarizing the current knowledge to clarify the dual role of reversine as a dedifferentiating agent and anti-cancer drug.

Bromodeoxyuridine (BrdU) marker for proliferating cells in living tissues: Detection in adult avian brain

Knowing how to perform cell proliferation assays is an undeniably essential skill to detect the effect of external or internal stimuli on cell genesis. Immunohistochemical staining using Bromodeoxyuridine (BrdU), enables quantification of proliferative fraction of cells in sectioned tissues. BrdU, an artificial nucleoside that substitutes for thymidine. It is currently quite popular research tool of choice for new cell genesis studies. It incorporates into the DNA during S-phase of the cell cycle that’s why used for birth dating and observing cell proliferation. BrdU immunohistochemistry is currently the most used technique for studying adult cell genesis in situ. In this article we provide a step-by step protocol for immunodetection of BrdU by light microscopy using avian species (Ploceus philippinus); starting from BrdU administration to the final analysis.