Multilineage potential of adult human mesenchymal stem cells.

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Antiviral effect and ex vivo CD4+ T cell proliferation in HIV-positive patients as a result of CD28 costimulation.

Because stimulation of CD4+ lymphocytes leads to activation of human immunodeficiency virus-type 1 (HIV-1) replication, viral spread, and cell death, adoptive CD4+ T cell therapy has not been possible. When antigen and CD28 receptors on cultured T cells were stimulated by monoclonal antibodies (mAbs) to CD3 and CD28 that had been immobilized, there was an increase in the number of polyclonal CD4+ T cells from HIV-infected donors. Activated cells predominantly secreted cytokines associated with T helper cell type 1 function. The HIV-1 viral load declined in the absence of antiretroviral agents. Moreover, CD28 stimulation of CD4+ T cells from uninfected donors rendered these cells highly resistant to HIV-1 infection. Immobilization of CD28 mAb was crucial to the development of HIV resistance, as cells stimulated with soluble CD28 mAb were highly susceptible to HIV infection. The CD28-mediated antiviral effect occurred early in the viral life cycle, before HIV-1 DNA integration. These data may facilitate immune reconstitution and gene therapy approaches in persons with HIV infection.

Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.

We determined that the defect in beta interferon induction in Vero cells is due to the absence of the simian beta interferon (IFN-beta) gene. Nevertheless, the human IFN-beta gene or a hybrid gene, in which the human IFN-beta promoter-regulatory region directs expression of the chloramphenicol acetyltransferase gene (pIFN-CAT), could be induced in transfected Vero cells, and these cells also regulated IFN-beta mRNA (but not pIFN-CAT mRNA) posttranscriptionally. These results indicate that the instability in the human IFN-beta gene is coded for by the coding or 3'-end region of IFN-beta mRNA and that the human IFN-beta gene is regulated in Vero and human cells in an identical manner.

Effect of human fibroblast interferon on the antiviral activity of mammalian cells treated with bleomycin, vincristine, or mitomycin C.

Bleomycin, vincristine, or mitomycin C, when added to HeLa cells simultaneously with human fibroblast interferon (IFN-beta), caused a decrease in cell density and inhibited DNA synthesis compared with HeLa cells treated with IFN-beta alone. However, the IFN-beta-induced antiviral processes were unaffected by the presence of these drugs as determined by in vitro enzyme assays and the development of the antiviral state in the intact HeLa cell. HeLa cells treated with IFN-beta alone or with IFN-beta in combination with bleomycin, vincristine, or mitomycin C were able to induce the double-stranded RNA-dependent adenosine triphosphate:2',5'-oligoadenylic acid adenyltransferase (EC 2.2.2.-) and the double-stranded RNA-dependent protein kinase. Furthermore, the antiviral state as measured by the reduction of plaque-forming units after infection of treated cells (with IFN-beta alone or with IFN-beta plus drugs) with vesicular stomatitis virus was not affected. These results indicate that, under these experimental conditions, the double-stranded RNA-dependent adenosine triphosphate:2',5'-oligoadenylic acid adenyltransferase and protein kinase can be induced by IFN-beta in cells treated with bleomycin, vincristine, or mitomycin C. These cells also develop the antiviral state. These experiments could provide a basis for a careful examination of the effects of interferon on the development of the antiviral state when testing potentially active antineoplastic agents. The possibility that IFN-beta potentiates the cytotoxic effects of bleomycin and mitomycin C on HeLa cells is also discussed.

RevM10-mediated inhibition of HIV-1 replication in chronically infected T cells.

Two clinical regimens have been proposed for gene therapies of acquired immunodeficiency syndrome (AIDS): (i) Genetic modification of differentiated peripheral mononuclear cells ex vivo and (ii) gene delivery into hematopoietic stem/progenitor cells ex vivo. Various antiviral strategies targeted at different molecular processes in the human immunodeficiency virus type 1 (HIV-1) life cycle are currently being pursued, all with the goal of reducing HIV-1 replication. Until now, all successful studies have reported inhibition in acutely HIV-infected cells that had been genetically modified prior to infection. These promising results do not address a clinically relevant question: What is the contribution of already infected peripheral mononuclear and hematopoietic stem/progenitor cells to disease progression? In this report, we demonstrate inhibition of both HIV-1 replication and production of infectious particles in chronically infected human T leukemia cell lines. The antiviral effect on the transduced cell population correlates with the expression of the dominant-negative RevM10 protein. This is the first demonstration that a gene therapy-based treatment can achieve antiviral efficacy in human T leukemia cells chronically infected with HIV-1.

Consequences of stable transduction and antigen-inducible expression of the human interleukin-7 gene on tetanus-toxoid-specific T cells.

Interleukin-7 (IL-7) has previously been shown to increase antigen-specific immune responses; the effect of IL-7 on human antigen-specific T cell lines has not directly been addressed. A tetanus-toxoid (TT)-specific T cell line exhibited increased proliferation in the presence of exogenous IL-7, suggesting that IL-7 may be useful in the potentiation of immune responses to defined microbial antigens. Murine retroviral vectors encoding the human IL-7 gene and the neomycin phosphotransferase gene (neoR) were packaged into murine retroviral particles, and supernatants containing these retroviral vectors were used to infect a CD4+ lymphoblastoid cell line. Stable integration of the retroviral vector and constitutive expression of the IL-7 gene were observed. Successful IL-7 gene transduction into TT-specific T cells was also accomplished. Detection of neoR DNA sequences and expression of IL-7-specific mRNA increased with selection in geneticin. Production of IL-7 in these cells was induced by exposure to TT. Production of IL-4, IL-6, and interferon-gamma (IFN-gamma) was detected after antigenic stimulation; there was, however, no effect of IL-7 on the pattern or kinetics of cytokine production by these cells. Human IL-7 transduced cells showed greater proliferation to TT than control T cells, particularly at subthreshold TT concentrations. These dta imply that genetic modification of antigen-specific T cells may be a plausible strategy for the study and manipulation of the immune responses to microbial pathogens.

Monoclonal antibodies reactive with human osteogenic cell surface antigens.

Monoclonal antibodies (McAbs) against the surface of osteoblastic cells have been used to characterize the osteogenic lineage. In view of the paucity of probes against the surface of normal human osteogenic cells, we sought to generate McAbs which could be used for both in vivo and in vitro studies. We raised a series of McAbs against early osteoblastic cell surface antigens by immunizing mice with human mesenchymal stem cells (MSCs) that had been directed into the osteogenic lineage in vitro. After screening against the surface of osteogenic cells at various stages of differentiation in vitro, as well as evaluating in situ reactivity with human fetal limbs, we isolated three hybridoma cell lines referred to as SB-10, SB-20, and SB-21. Immunocytochemical analyses during osteogenic differentiation demonstrate that SB-10 reacts with MSCs and osteoprogenitors, but no longer reacts with cells once alkaline phosphatase (APase) is expressed. Flow cytometry documents that SB-10 is expressed on the surface of all purified, culture-expanded human MSCs, thus providing further evidence that these cells are a homogeneous population. By contrast, SB-20 and SB-21 do not react with the progenitor cells in situ, but bind to a subset of the APase-positive osteoblasts. None of these antibodies stain terminally differentiated osteocytes in sections of developing bone. Furthermore, these McAbs were not observed to react in samples from chick, rat, rabbit, canine, or bovine bone, although selected extraskeletal human tissues were immunostained. In all cell and tissue specimens examined, SB-20 immunostaining is identical to that observed with SB-21. We have used these McAbs to refine our understanding of the discrete cellular transitions that constitute the osteogenic cell lineage. We suggest a refined model for understanding osteoblast differentiation that is based on the proposition that the sequential acquisition and loss of specific cell surface molecules can be used to define positions of individual cells within the osteogenic cell lineage.

Consequences of human immunodeficiency virus type 1 superinfection of chronically infected cells.

Infection of T cell lines by the type 1 human immunodeficiency virus (HIV-1) is associated with downregulation of the CD4 receptor and resistance to further HIV-1 infection, the phenomenon of viral interference. The ACH2 cell line, a model for chronic HIV-1 infection, possesses a single integrated copy of the HIV-1 strain LAI, is essentially CD4 negative, and can be induced to make virus by a variety of stimuli. We utilized the known sequence differences between HIVLAI and HIVRF to devise a polymerase chain reaction (PCR) strategy that permits reliable and quantitative discrimination between the two strains. We demonstrate that ACH2 cells can be superinfected by HIVRF at a frequency of 60-300 HIVRF genomes/10(4) ACH2 cells and that the frequency of superinfection appears to increase with time. Reverse transcription of ACH2 mRNA from days 13, 27, and 38 postinfection allowed a similar PCR strategy (RT-PCR) to be used to analyze full-length HIVRF- and HIVLAI-specific transcripts. These data suggested that superinfection of ACH2 with HIVRF results in an increase in expression of both HIVRF and HIVLAI mRNA. From day 13 to day 38 postinfection there was an increase in the relative expression of HIVRF compared with HIVLAI. By day 38, when only 1.1% of HIV DNA sequences were HIVRF derived, roughly 80% of the HIV-specific full-length mRNA was HIVRF in origin, with a concomitant decrease in HIVLAI transcription.

Providing a Microenvironment for the Development of Human CD34+ Hematopoietic Cells in SCID Mice.

In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11-10). The Lof(11-10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11-10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11-10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11-10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed. Copyright 1997 S. Karger AG, Basel

Characterization of a Human Stromal Cell Line Supporting Hematopoietic Progenitor Cell Proliferation: Effect of HIV Expression.

Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from human bone marrow by transfecting a plasmid containing the SV40 large T-antigen and isolating foci exhibiting a transformed phenotype. The Lof(11-10) cell line was characterized to determine its susceptibility to HIV infection, to identify its cytokine production profile, and to test the ability of conditioned media from this line to support CD34+ cell differentiation in the presence and absence of HIV expression. Nine cytokines were detected by RT-PCR and ELISA analysis. Conditioned media obtained from the Lof(11-10) cell line was able to support CD34+ celle differentiation. However, because the Lof(11-10) cells are not infectible by HIV, molecular clones of HIV were introduced into these cells by transfection. There was no qualitative difference in the levels of cytokine production between HIV-expressing and control Lof(11-10) cells. Furthermore, conditioned media derived from HIV-expressing and control Lof(11-10) cells added to bone marrow-derived CD34+ progenitor cells yielded similar colony formation in methylcellulose assays. Our data suggest that HIV infection of the cytokine-producing cells within the bone marrow microenvironment, as represented by the Lof(11-10) cell line, results in both normal cytokine production and hematopoiesis in spite of HIV expression. This report adds to the evidence against stromal cells being a significant target of HIV and establishes a system for comparison with more relevant models. Copyright 1995 S. Karger AG, Basel

Human mesenchymal stem cells respond to fibroblast growth factors.

Human mesenchymal stem cells can be isolated from bone marrow aspirates, purified and cultured for many passages without losing their unique properties. One of the hallmarks of stem cells is pluripotency, and human mesenchymal stem cells can be induced to assume phenotypes of mesenchymal tissues including, but not limited to, those of osteocytes, chondrocytes and adipocytes. Due to their ability to form cartilage, bone, fat and other connective tissue, human mesenchymal stem cells have great potential in regenerating diseased or injured tissues. Successful growth of human mesenchymal stem cells is essential to this process, and we have examined the response of human mesenchymal stem cells towards FGF1 and FGF2, two potent growth factors for human tissues. We provide evidence that: 1) human mesenchymal stem cells produce mRNA for receptors for FGF1 and FGF2; 2) these receptors can be detected on the surface of human mesenchymal stem cells; 3) FGF1 and FGF2 increase the rate at which human mesenchymal stem cells proliferate.

Methylation as a modulator of expression of human immunodeficiency virus.

When Vero or murine cells were stably transfected with the human immunodeficiency virus (HIV) long terminal repeat (LTR) that directs the chloramphenicol acetyltransferase (CAT) gene (pU3R-III-CAT), expression was suppressed. Treatment with the nucleoside analog 5-azacytidine (5-azaC) restored CAT expression. S1 nuclease analysis and a nuclear run-on assay demonstrated that activation of the latent HIV LTR by 5-azacytidine occurred at the transcriptional level. Southern blot analysis demonstrated that this activation was due to the demethylation of cytosine residues in the LTR enhancer. Thus, the HIV LTR appears to be susceptible to transcriptional inactivation by methylation, a process that is proposed to play a modulatory role in viral latency.

Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism.

To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is reporter gene specific and promoter independent.

Inhibition of human immunodeficiency virus (HIV) replication by HIV-trans-activated alpha 2-interferon.

We have prepared stable cell lines, derived from Vero cells and A3.01 cells, that express a hybrid human alpha 2-interferon gene under control of the human immunodeficiency virus (HIV) long terminal repeat. These cells constitutively produced low levels (50-150 units/ml) of alpha 2-interferon. However, high levels of interferon (10(3) units/ml) could be induced upon trans-activation by the product of the tat gene (pIIIextatIII), and de novo infection by HIV resulted in a moderate increase (400 units/ml) in alpha 2-interferon synthesis. In contrast to the fully permissive HIV replication, in transfected Vero cells or infected A3.01 cells, the transcription and replication of HIV in Vero or A3.01 cells containing the HIV long terminal repeat--alpha 2-interferon hybrid gene (VN89 and A3N89 cells, respectively) was completely inhibited. These data suggest that virus-trans-activated alpha 2-interferon synthesis can be used as a selective inhibitor of HIV replication.

Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes.

Stable DNA-transfected Vero cell lines that express the major immediate-early nuclear antigen (IE68) of HCMV-(Towne) have been established. Immunofluorescence staining with monoclonal antibodies revealed that the protein was distributed either in a uniform diffuse nuclear pattern or as punctate nuclear granules in up to 80% of the cells in these cultures. In addition, 1 to 2% of the positive nuclei gave a distinctive staining pattern suggesting an association with the chromosomes of mitotic cells. Colcemid-blocking studies confirmed that most of the IE antigen was localized in the vicinity of condensed chromosomes in all metaphase cells after methanol fixation. In contrast, the SV40 large T-antigen protein was found to be preferentially excluded from metaphase chromosomes in a similar colcemid-treated human cell line. In transient expression assays, 1 to 2% of IE antigen-positive Vero, 293, or Balb/c3T3 cells also displayed a metaphase chromosome association pattern. Mapping studies using deletion and truncation mutants revealed that the monoclonal antibodies recognized epitopes encoded within the small NH2-terminal exons that are common to both the IE1 and IE2 gene products. However, an intact exon-4 (IE1) region, but not the exon-5 (IE2) region of the HCMV IE gene complex, was required for conferring both the normal diffuse nuclear localization pattern and the chromosome-association properties. Furthermore, removal of the glutamic acid-rich COOH-terminal coding portions of exon-4 resulted in aberrant staining patterns with production of large, phase-dense nuclear globules in all positive cells. An association between the IE68 IE1 protein and metaphase chromosomes was also detected after HCMV-(Towne) infection in a small proportion of both nonpermissive Balb/c3T3 cells and permissive HF cells. We conclude that the IE1 acidic nuclear phosphoprotein displays some properties similar to those of the EBNA-1 protein of Epstein-Barr virus and suggest that it may potentially play a role in maintenance of the latent state of HCMV DNA.

Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines.

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.

Novel induction by herpes simplex virus of hybrid interferon gene transcripts driven by the strong cytomegalovirus IE94 promoter.

We have constructed stable DNA-transfected LTK+ cell lines containing two different coselected hybrid interferon (IFN) genes driven by the usually strong and constitutive promoter from the immediate-early 94K protein (IE94) gene of simian cytomegalovirus. Surprisingly, and unlike hybrid IE94-chloramphenicol acetyltransferase gene constructs, both of the IE94-IFN genes (one with and one without the complex spliced intron region) produced relatively low basal titers of biologically active human IFN in the mouse cell lines. However, IFN expression could be stimulated up to 120-fold by superinfection with herpes simplex virus (HSV), although not with cytomegalovirus. To examine the mechanism of this unexpected HSV induction process, we measured the levels of both IE94-IFN mRNA and IFN protein produced under various infection protocols. Compared with similar previously characterized cell lines containing hybrid IFN genes under the control of HSV IE or delayed-early (DE) promoters, activation of IFN expression first occurred at an intermediate time. Both IE94-IFN cell lines also produced an unusual pattern of response to infection with the HSV IE regulation-deficient mutants tsK and tsB7: stimulation of IFN synthesis occurred in the absence of a functional HSV IE175 (or ICP4) gene product, but did not occur in the absence of uncoating of virus capsids. Cycloheximide treatment (without virus infection) also gave a rapid 30-fold increase in steady-state levels of correctly initiated mRNA from both types of IE94-IFN hybrid genes, but had no effect on cells containing the IE175-IFN construct. Therefore, we conclude that the use of the IE94-IFN constructs identifies a novel HSV regulatory response that requires a previously unrecognized function of HSV and does not involve either IE175 or the pre-IE "virion factor" trans-activators that are known to stimulate transcription of HSV IE and DE genes, respectively.

Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells.

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1alpha induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1alpha induced IL-1alpha and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34+ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment.