Todd Lenvik

Pluripotency of mesenchymal stem cells derived from adult marrow

We report here that cells co-purifying with mesenchymal stem cells—termed here multipotent adult progenitor cells or MAPCs—differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17)

The Fc receptor CD16 is present on essentially all CD56(dim) peripheral blood natural killer (NK) cells. Upon recognition of antibody-coated cells it delivers a potent signal to NK cells, which eliminate targets through direct killing and cytokine production. Here we investigated the regulation of CD16 surface expression after NK cell activation. Cytokine activation and target cell stimulation led to marked decreases in CD16 expression. Activation of CD56(dim) NK cells by cross-linking CD16 with antibodies resulted in a loss of CD16 and CD62L, which correlated with increased interferon-γ production. A disintegrin and metalloprotease-17 (ADAM17) is shown to be expressed by NK cells, and its selective inhibition abrogated CD16 and CD62L shedding, and led to enhanced interferon-γ production, especially when triggering was delivered through CD16. Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets was also enhanced by ADAM17 inhibition. This supports an important role for targeting ADAM17 to prevent CD16 shedding and improve the efficacy of therapeutic antibodies. Our findings demonstrate that over-activation of ADAM17 in NK cells may be detrimental to their effector functions by down-regulating surface expression of CD16 and CD62L.

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

Cutting Edge: KIR Antisense Transcripts Are Processed into a 28-Base PIWI-Like RNA in Human NK Cells

Killer Ig-like receptors (KIRs) are expressed in a variegated, clonally restricted fashion on NK cells and are important determinants of NK cell function. Although silencing of individual KIR genes is strongly correlated with the presence of CpG dinucleotide methylation within the promoter, the mechanism responsible for silencing has not been identified. Our results show that antisense transcripts mediate KIR transcriptional silencing through a novel PIWI-like 28-base small RNA. Although PIWI RNA-mediated silencing of transposable elements within germ cells have been described, this is the first report that identifies a PIWI-like RNA in an immune somatic cell lineage and identifies a mechanism that may be broadly used in orchestrating immune development.

Erratum: Pluripotency of mesenchymal stem cells derived from adult marrow (Nature (2002) 418 (41-49) DOI: 10.1038/nature00870)

This corrects the article DOI: 10.1038/nature00870

Chlamydomonas transcripts encoding three divergent plastid chaperonins are heat-inducible

Three cDNAs encoding plastid cpn60 chaperonin subunits have been isolated from the unicellular green alga Chlamydomonas reinhardtii. Based on comparisons of the predicted amino acid sequences, we conclude that Chlamydomonas, like higher plants, contains divergent plastid cpn60-α and cpn60-β subunits. The predicted amino acid sequences of the two Chlamydomonas cpn60-β subunits differ significantly (24% divergent), indicating that the two cpn60-β subunits have been selectively maintained for a considerable period of time. Unlike plastid chaperonin trnascripts in higher plants, heat shock conditions (42°C) lead to a rapid increase (10-to 30-fold) in the level of each of the three plastid transcripts.

Transcriptional regulation of Munc13-4 expression in cytotoxic lymphocytes is disrupted by an intronic mutation associated with a primary immunodeficiency

A conserved regulatory element in intron 1 of UNC13D regulates Munc13-4 expression.

Identification of a KIR antisense lncRNA expressed by progenitor cells

Human NK cells express cell surface class I MHC receptors (killer cell immunoglobulin-like receptor, KIR) in a probabilistic manner. Previous studies have shown that a distal promoter acts in conjunction with a proximal bidirectional promoter to control the selective activation of KIR genes. We report here the presence of an intron 2 promoter in several KIR genes that produce a spliced antisense transcript. This long noncoding RNA (lncRNA) transcript contains antisense sequence complementary to KIR-coding exons 1 and 2 as well as the proximal promoter region of the KIR genes. The antisense promoter contains myeloid zinc finger 1 (MZF-1)-binding sites, a transcription factor found in hematopoietic progenitors and myeloid precursors. The KIR antisense lncRNA was detected only in progenitor cells or pluripotent cell lines, suggesting a function that is specific for stem cells. Overexpression of MZF-1 in developing NK cells led to decreased KIR expression, consistent with a role for the KIR antisense lncRNA in silencing KIR gene expression early in development.

Characterization of rps17, rpl9 and rpl15: three nucleus-encoded plastid ribosomal protein genes

Approximately two-thirds of the 55 to 60 plastid ribosomal proteins are encoded in the nucleus. Since the protein products of each of these genes are needed in equal amounts for ribosome assembly, their expression may be coordinately regulated by common mechanisms. To begin to understand how the expression of these genes is regulated, we have isolated cDNA and genomic clones for three plastid ribosomal protein genes from an Arabidopsis thaliana library. The genes rps17, rpl9 and rpl15, encoding plastid ribosomal proteins CS17, CL9 and CL15, respectively, are located in the nuclear genome and Southern blot data suggest that each is a single copy gene in A. thaliana. Northern blot data show that transcripts from rps17, rpl9 and rpl15 are much more abundant in leaves and stems than they are in roots. The nucleotide sequences of each of these three genes were determined and their transcriptional initiation sites identified. rps17 transcripts have multiple 5′ ends suggesting that they are initiated at multiple sites or are post-transcriptionally processed at their 5′ end. rpl9 and rpl15 apparently have unique transcriptional initiation sites but are post-transcriptionally processed to remove six and three introns, respectively, from their primary transcripts. We have examined the genomic sequences for motifs that may be important for the proper expression of these genes. A 7 bp sequence motif, whose consensus is 5′-AGGCCCA-3′, flanked by AT-rich regions was identified between 38 and 73 nucleotides upstream of the rps17, rpl9 and rpl15 transcriptional initiation sites.