Michel Revel

HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34 + stem cell recruitment to the liver

Hematopoietic stem cells rarely contribute to hepatic regeneration, however, the mechanisms governing their homing to the liver, which is a crucial first step, are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1), which attracts human and murine progenitors, is expressed by liver bile duct epithelium. Neutralization of the SDF-1 receptor CXCR4 abolished homing and engraftment of the murine liver by human CD34+ hematopoietic progenitors, while local injection of human SDF-1 increased their homing. Engrafted human cells were localized in clusters surrounding the bile ducts, in close proximity to SDF-1-expressing epithelial cells, and differentiated into albumin-producing cells. Irradiation or inflammation increased SDF-1 levels and hepatic injury induced MMP-9 activity, leading to both increased CXCR4 expression and SDF-1-mediated recruitment of hematopoietic progenitors to the liver. Unexpectedly, HGF, which is increased following liver injury, promoted protrusion formation, CXCR4 upregulation, and SDF-1-mediated directional migration by human CD34+ progenitors, and synergized with stem cell factor. Thus, stress-induced signals, such as increased expression of SDF-1, MMP-9, and HGF, recruit human CD34+ progenitors with hematopoietic and/or hepatic-like potential to the liver of NOD/SCID mice. Our results suggest the potential of hematopoietic CD34+/CXCR4+cells to respond to stress signals from nonhematopoietic injured organs as an important mechanism for tissue targeting and repair.

Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines.

Induced human fibroblasts produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3‐kb RNA designated IFN‐beta 2 and distinct from the 0.9‐kb IFN‐beta 1 mRNA. In vitro transcription‐‐translation mapping of the full‐length IFN‐beta 2 cDNA sequence, shows that it encodes a 23.7‐kd protein of 212 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN‐beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN‐beta 2. Specific immunoassays show that IFN‐beta 2 secreted by DNA‐transformed rodent cells is a processed 21‐kd protein, whose activity is cross‐neutralized by antibodies to human IFN‐beta 1 but not to IFN‐alpha or gamma. The immunoassay also demonstrates the induction of IFN‐beta 2 secretion by fibroblasts in response to growth‐regulatory cytokines, such as interleukin‐1 and tumor necrosis factor. The function of this IFN‐beta 2 as an autoregulatory inhibitor of cell growth is discussed.

Interferon-activated genes

Abstract Interferons (IFNs) are cell-secreted protein factors which are not only involved in the defence against virus infections, but play a key role in the regulation of cell growth and differentiation. More than 10 cellular genes have now been identified as being specifically induced by IFNs and hence possible mediators of the complex biological action of IFNs. Transcription of several of these genes is activated within minutes following interaction of IFNs with their cell surface receptors.

A protein-arginine methyltransferase binds to the intracytoplasmic domain of the IFNAR1 chain in the type I interferon receptor

The intracytoplasmic domain (IC) of cytokine receptors provides docking sites for proteins which mediate signal transduction. Thus, in interferon‐α,β receptors (IFNAR1 and 2), the IC region binds protein‐tyrosine and ‐serine/threonine kinases which phosphorylate the receptor and the associated Stat transcription factors. A two‐hybrid screening was carried out to identify additional proteins which could interact with the IC domain of the IFNAR1 chain of the IFN‐α,β receptor. Several positive clones representing a protein sequence designated IR1B4 were recovered from a human cDNA library. IR1B4 was identified as the human homolog of PRMT1, a protein‐arginine methyltransferase from rat cells. Flag‐IR1B4 fusion proteins bind to the isolated IFNAR1 intracytoplasmic domain produced in Escherichia coli, as well as to the intact IFNAR1 chain extracted by detergent from human U266 cell membranes. S‐Adenosylmethionine‐dependent methyltransferase activity was precipitated by anti‐IFNAR1 antibodies from untreated human cells. IR1B4/PRMT1 is involved in IFN action since U266 cells rendered deficient in this methyltransferase by antisense oligonucleotides become more resistant to growth inhibition by IFN. Methylation of proteins by enzymes which can attach to the IC domains of receptors may be a signaling mechanism complementing protein phosphorylation. Among substrates methylated by PRMT1 are RNA‐binding heterogeneous nuclear ribonucleoproteins (hnRNPs) which are involved in mRNA processing, splicing and transport into the cytoplasm.

Specific phosphorylation in vitro of a protein associated with ribosomes of interferon-treated mouse L cells.

Cultures of L cells treated with mouse interferon yield cell extracts with a reduced activity to translate exogenously added mRNAs into proteins [l-5] . There is a strong correlation between the interferoninduced antiviral state in intact cells and the development of the in vitro translational inhibition [3] . The selectivity of interferon’s effect against viral, but not cellular, protein synthesis in intact cells [6-81 is, however, partially lost in the cell-free systems at high doses of interferon [3 ] . The reduced translational activity in vitro results from a dominant inhibitor(s), loosely associated with the ribosomes, whose activity increases with the dose of interferon used to treat the cells [3], but which has been only partially purified and characterized [3-5,9]. The inhibition affects both initiation and elongation of the polypeptide chains [9--l 11, and can be reduced by supplementing the extracts with excess amounts of some tRNA species [ II151. In contrast, the addition of double stranded RNA (ds RNA), as poly I: C [ 161 or the replicative form of Mengo RNA [ 111, increases the translation inhibition seen in extracts of interferon-treated cells. Recent work with reticulocyte lysates [ 17191 has indicated that the inhibitory activity of ds RNA on translation may be mediated by protein kinase activities. This observation prompted us to examine whether the increased sensitivity of extracts from interferon treated cells to ds RNA, is also mediated by protein phosphorylation. We show, here, that in the subcellular fraction which contains the interferoninduced inhibitor(s) of translation, there is strong

Structure of two forms of the interferon-induced (2'-5') oligo A synthetase of human cells based on cDNAs and gene sequences.

The (2′‐5′) oligo A synthetase E, one of the translational inhibitory enzymes whose synthesis is strongly induced by all interferons (IFNs), is shown to be encoded in human cells by a 13.5‐kb gene. By a cell‐specific differential splicing, between the seventh and an additional eighth exon of this gene, two active E mRNAs of 1.6 and 1.8 kb are produced, along with several longer transcripts. cDNA clones for the two mRNAs were obtained and their sequences indicate that the human (2′‐5′) oligo A synthetase gene codes for two forms of the enzyme of mol. wt. 41 000 and 46 000, which differ only by their C‐terminal ends. The product of the 1.6‐kb RNA (E16) has a very hydrophobic C terminus, which is replaced by a longer acidic C‐terminal sequence in the 1.8‐kb RNA product (E18). The transcriptional start site of the gene was identified and 200 bp of the 5′ flanking region were sequenced. A strong homology was found between this region of the IFN‐activated (2′‐5′) oligo A synthetase gene and the corresponding region of the human fibroblast IFN‐beta 1 gene, whose transcription is also stimulated by IFN priming. The gene has two polyadenylation sites which share a common undecanucleotide, but are used in a cell‐specific manner to give rise to the 1.6‐ and 1.8‐kb mRNAs.

Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo.

In attempts to produce mature oligodendrocytes from human embryonic stem (huES) cells, we searched conditions inducing transcription factors Olig1/2, as well as Nkx2.2 and Sox10, which are needed for maturation. This was obtained by retinoic acid treatment followed by noggin, an antagonist of bone morphogenetic proteins (BMPs). We found that retinoic acid induces BMPs in huES cells. Addition of noggin at a specific step was essential to form numerous mature oligodendrocytes with ramified branches and producing myelin basic protein (MBP). We describe a procedure converting huES cells into enriched populations of oligodendrocyte precursors that can be expanded and passaged repeatedly and subsequently differentiated into mature cells. Transplantation of such precursors showed that pretreatment by noggin markedly stimulates their capacity to myelinate in the brain of MBP-deficient shiverer mice in organotypic cultures and in living animals. Arrays of numerous long MBP+ fibers were generated over extended areas in the brain, with evidence of cell migration after transplantation and with formation of compact myelin sheaths.

Differential tyrosine phosphorylation of the IFNAR chain of the type I interferon receptor and of an associated surface protein in response to IFN-alpha and IFN-beta.

The human interferon alpha‐receptor (IFNAR gene product) is a transmembranal protein of 557 amino acids with an intracytoplasmic domain of 100 amino acids containing four tyrosines. Antibodies to a C‐terminal peptide (residues 521‐536) were developed which efficiently immunoprecipitate the 105 kDa IFNAR protein from detergent extracts of human cells. We show that the IFNAR protein becomes tyrosine phosphorylated within 5 min after treatment of human myeloma U266 cells with IFN‐alpha 2, IFN‐alpha 8 or IFN‐beta. The IFNAR chain interacts with both IFN‐alpha 2 and IFN‐beta, as demonstrated by cross‐linking. Among elements involved in signal transduction by type I IFNs, the tyrosine kinase Tyk2 but not Jak1, and the ISGF3 transcription factor subunit Stat2 (p113) but not Stat1 (p91), are found associated with the IFNAR protein. After IFN‐beta treatment for 5 min, a tyrosine‐phosphorylated protein of approximately 95 kDa (beta‐PTyr) is found bound to IFNAR, but can be dissociated by denaturation. The beta‐PTyr protein is present on the cell surface, like IFNAR, as shown by extracellular biotin tagging. The ratio of beta‐PTyr to IFNAR tyrosine phosphorylation is much higher with IFN‐beta than with IFN‐alpha 2 or 8. Both are IFN dependent and abrogated by a monoclonal antibody which blocks IFNAR action. The beta‐PTyr component may represent an important difference in the action of IFN‐beta as compared with IFN‐alpha in their shared receptor system.

Interferon action: isolation of nuclease F, a translation inhibitor activated by interferon-induced (2'-5') oligo-isoadenylate.

Cell cultures treated by interferon become unable to support viral replication. At least one of interferon’s antiviral effects is to inhibit viral protein synthesis [l-3]. Interferon induces several biochemical mechanisms which could mediate this inhibition: (i) A double-stranded RNA (dsRNA) and ATP-dependent phosphorylation of initiation factor eIF2 and possibly other ribosome-associated proteins [4-71; (ii) The dsRNA ATP-dependent synthesis of an unusual oligo-adenylate isomer with 2’-5 phosphodiester bonds [7-91; (iii) A mechanism which affects polypeptide chain elongation, that does not require dsRNA and is reversed by tRNAs [lo-l 21. We have reported [3,9] the separation and isolation, from extracts of interferon-treated L cells, of the dsRNA ATP-activated protein kinase PKi, and of the (2’-5’) oligo-isoadenylate synthetase E. Here, we demonstrate that the oligonucleotide produced by E requires, to inhibit mRNA translation, an additional protein F already present constitutively in untreated cells. The purified F is shown to be an oligo-isoadenylate-dependent ribonuclease which degrades the mRNA template. Our data show that the dsRNA ATP-dependent nuclease activity reported [ 13,141 to be increased in extracts of interferon-treated cells, can be explained as the activation of a constitutive ribonuclease, F, by the product of the interferon-induced and dsRNA-dependent oligo-isoadenylate synthetase E. This is in

Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells

Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.