Alice Pébay

Essential Roles of Sphingosine‐1‐Phosphate and Platelet‐Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum—sphingosine‐1‐phosphate (S1P), lysophosphatidic acid (LPA), and platelet‐derived growth factor (PDGF)—in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum‐free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri‐potentiality.

Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades

Sphingosine‐1‐phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg‐1 and Edg‐3. S1P stimulated thymidine incorporation and induced activation of extracellular signal‐regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P‐evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3‐kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin‐sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood–brain barrier.

Generation of Induced Pluripotent Stem Cell Lines from Friedreich Ataxia Patients

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibroblasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results in the expression of frataxin protein in differentiated neurons. As such, iPS cell lines derived from FRDA patients, following correction of the mutated gene, could provide a useful source of immunocompatible cells for transplantation therapy.

Presence of functional gap junctions in human embryonic stem cells.

Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription—polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal‐regulated kinase inhibition.

Lysophosphatidic Acid Inhibits Neuronal Differentiation of Neural Stem/Progenitor Cells Derived from Human Embryonic Stem Cells

Lysophospholipids are signaling molecules that play broad and major roles within the nervous system during both early development and neural injury. We used neural differentiation of human embryonic stem cells (hESC) as an in vitro model to examine the specific effects of lysophosphatidic acid (LPA) at various stages of neural development, from neural induction to mature neurons and glia. We report that LPA inhibits neurosphere formation and the differentiation of neural stem cells (NSC) toward neurons, without modifying NSC proliferation, apoptosis, or astrocytic differentiation. LPA acts through the activation of the Rho/ROCK and the phosphatidylinositol 3‐kinase/Akt pathways to inhibit neuronal differentiation. This study is the first demonstration of a role for LPA signaling in neuronal differentiation of hESC. As LPA concentrations increase during inflammation, the inhibition of neuronal differentiation by LPA might contribute to the low level of neurogenesis observed following neurotrauma.

Role of Gap Junctions in Embryonic and Somatic Stem Cells

Stem cells provide an invaluable tool to develop cell replacement therapies for a range of serious disorders caused by cell damage or degeneration. Much research in the field is focused on the identification of signals that either maintain stem cell pluripotency or direct their differentiation. Understanding how stem cells communicate within their microenvironment is essential to achieve their therapeutic potentials. Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ES cells) and various somatic stem cells. GJIC has been implicated in regulating different biological events in many stem cells, including cell proliferation, differentiation and apoptosis. This review summarizes the current understanding of gap junctions in both embryonic and somatic stem cells, as well as their potential role in growth control and cellular differentiation.

Small-Molecule Induction of Neural Crest-like Cells Derived from Human Neural Progenitors†‡§

Neural crest (NC) cells are stem cells that are specified within the embryonic neuroectodermal epithelium and migrate to stereotyped peripheral sites for differentiation into many cell types. Several neurocristopathies involve a deficit of NC‐derived cells, raising the possibility of stem cell therapy. In Hirschsprungs disease the distal bowel lacks an enteric nervous system caused by a failure of colonization by NC‐derived cells. We have developed a robust method of producing migrating NC‐like cells from human embryonic stem cell–derived neural progenitors using a coculture system of mouse embryonic fibroblasts. Significantly, subsequent exposure to Y27632, a small‐molecule inhibitor of the Rho effectors ROCKI/II, dramatically increased the efficiency of differentiation into NC‐like cells, identified by marker expression in vitro. NC‐like cells derived by this method were able to migrate along NC pathways in avian embryos in ovo and within explants of murine bowel, and to differentiate into cells with neuronal and glial markers. This is the first study to report the use of a small molecule to induce cells with NC characteristics from embryonic stem cells that can migrate and generate neurons and support cells in complex tissue. Furthermore, this study demonstrates that small‐molecule regulators of ROCKI/II signaling may be valuable tools for stem cell research aimed at treatment of neurocristopathies. STEM CELLS 2009;27:2896–2905

Regulation of stem cell pluripotency and neural differentiation by lysophospholipids.

Lysophospholipids are bioactive signalling molecules able to act through the binding of their specific G-protein-coupled receptors to exert pleiotropic effects on a wide range of cells. The most widely studied signalling lysophospholipids are lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). LPA and S1P have been identified to have widespread developmental, physiological and pathological actions in the central nervous system and more recently have been shown to induce biological effects on various stem cell types. This review aims to summarise the current knowledge on LPA and S1P regulation of embryonic and neural stem cell biology.

Fgf2 improves functional recovery—decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury

A major impediment for recovery after mammalian spinal cord injury (SCI) is the glial scar formed by proliferating reactive astrocytes. Finding factors that may reduce glial scarring, increase neuronal survival, and promote neurite outgrowth are of major importance for improving the outcome after SCI. Exogenous fibroblast growth factor (Fgf) has been shown to decrease injury volume and improve functional outcome; however, the mechanisms by which this is mediated are still largely unknown.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo

PURPOSE Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.