Human mesenchymal stem cells maintain transgene expression during expansion and differentiation.

Human adult bone marrow contains both hematopoietic stem cells that generate cells of all hematopoietic lineages and human mesenchymal stem cells (hMSCs), which support hematopoiesis and contribute to the regeneration of multiple connective tissues. The goal of the current study was to demonstrate that transduced hMSCs maintain transgene expression after stem cell differentiation in vitro and in vivo. We have introduced genes into cultured hMSCs by retroviral vector transfer and demonstrated long-term in vitro and in vivo expression of human interleukin 3 (hIL-3) and green fluorescent protein (GFP). Protocols were developed to achieve transduction efficiencies of 80-90% in these stem cells. In vitro expression of hIL-3 averaged 350 ng/10(6)cells/24 h over 17 passages (> 6 months) and GFP expression was stable over the same time period. Transduced hMSCs were able to differentiate into osteogenic, adipogenic, and chondrogenic lineages and maintained transgene expression after differentiation. Parallel studies were performed in vivo using NOD/SCID mice. Human MSCs expressing hIL-3 were cultured on several matrices and then delivered by subcutaneous, intravenous, and intraperitoneal routes. Sampling of peripheral blood demonstrated that systemic hIL-3 expression was maintained in the range of 100-800 pg/ml over a period of 3 months. These results illustrate the ability of hMSCs to express genes of therapeutic potential and demonstrate their potential clinical utility as cellular vehicles for systemic gene delivery.

Mesenchymal stem cells as vehicles for gene delivery.

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbon-ape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.

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.

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.

Mesenchymal stem cells in osteobiology and applied bone regeneration.

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. These cells, referred to as MSCs, can be purified and culture expanded from animals and humans. This review summarizes recent experimentation focused on characterizing the cellular aspects of osteogenic differentiation, and exploration of the potential for using autologous stem cell therapy to augment bone repair and regeneration. The authors have completed an array of preclinical studies showing the feasibility and efficacy of MSC based implants to heal large osseous defects. After confirming that syngeneic rat MSCs could heal a critical size segmental defect in the femur, it was established that human MSCs form bone of considerable mechanical integrity when implanted in an osseous defect in an immunocompromised animal. Furthermore, bone repair studies in dogs verify that the technology is transferable to large animals, and that the application of this technology to patients at geographically remote sites is feasible. These studies suggest that by combining MSCs with an appropriate delivery vehicle, it may be possible to offer patients new therapeutic options.

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.

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.

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

Transcriptional effects of superinfection in HIV chronically infected T cells: studies in dually infected clones.

We had previously shown that chronically infected ACH-2 cells (HIVLAI) could be superinfected with HIVRF, that the frequency of superinfection increased with time, and that the transcription of the superinfecting virus exceeded that of the host HIVLAI provirus. In contrast, ACH-2 cells superinfected with a nef-substituted neomycin-resistant (proNEO) provirus were not detectable by DNA polymerase chain reaction (PCR) until geneticin (G418) was added, suggesting that the ability to propagate progressively in culture may be HIV strain specific. Clonal populations of ACH-2 superinfected with proNEO did not demonstrate preferential transcription of the superinfecting virus. However, clones of ACH-2 superinfected with HIVRF (ACH2/RF) showed a preponderance of HIVRF transcripts similar to that seen in bulk populations. Induction of the superinfecting virus by phorbol ester (PMA) occurred more rapidly than the hose provirus and did not equalize transcriptional activity. PCR-derived long terminal repeat (LTR) fragments and Tat cDNAs from A3.01 cells acutely infected with HIVRF or from ACH-2 cells were sequenced and tested for transactivation. The HIVLAI LTR was two to three times more Tat-responsive than the HIVRF LTR. TatRF was two to three times more transcriptionally active on either LTR than TatLAI. Demethylation with 5-azacytidine did not significantly affect HIV expression from the HIVLAI host provirus of superinfected ACH2/RF cell clones. These data suggest that the mechanism of preferential transcription in HIVRF superinfected ACH2/RF may be attributed to the Tat/TAR axis and the effect of the specific locus of host proviral integration.

Inhibition of HIV replication by sense and antisense rev response elements in HIV-based retroviral vectors.

The life cycle of human immunodeficiency virus type 1 (HIV-1) is critically dependent on the transregulatory proteins Tat and Rev. Tat increases the production of HIV-specific mRNAs by direct binding to the transactivation response (TAR) element located at the 5' end of all HIV transcripts. In contrast, Rev uses a complex RNA stem loop structure, the Rev response element (RRE), which is found in full-length and singly spliced HIV transcripts. Rev is required for the cytoplasmic expression of full-length mRNAs encoding Gag, Pol, and Env structural proteins. The complex intracellular interactions between Tat, Rev, host cell factors, and their respective RNA response elements should be susceptible to interdiction by genetic therapies designed to introduce and express novel genetic information. We show that the expression of antisense RREs inhibited the cytoplasmic expression of RRE containing HIV-1 transcripts. HIV-based retroviral vectors containing either the antisense (-) or sense (+) RREs inhibited HIV replication in transient transfections. The production of full-length HIV mRNA was also decreased significantly by the expression of RREs in either orientation. Interestingly, there was a paradoxic increase in HIV p24 gag production at low levels of inhibitor; this effect may have been the result of encapsidation of RRE-containing HIV-based retroviral vectors. The data suggest that the introduction and inducible expression of RRE-containing, HIV-based retroviral vectors may have therapeutic value in HIV infection.

Exposure of human CD34+ cells to human immunodeficiency virus type 1 does not influence their expansion and proliferation of hematopoietic progenitors in vitro.

The susceptibility of highly purified human CD34+ cells to monocytotropic (Ba-L) and lymphotropic (A018-post) strains of human immunodeficiency virus-1 (HIV-1) was examined. Liquid cultures initiated with fresh immunomagnetically purified CD34+ cells using the K6.1 CD34 monoclonal antibody (MoAb) (K6.1/CD34+) were positive for HIV expression 2 weeks after exposure to HIV-1 Ba-L. These cells were initially greater than 90% CD34+ and had undetectable monocyte contamination by flow-cytometric staining and side-scatter analyses, respectively, and undetectable T-cell contamination by CD3 polymerase chain reaction (PCR) analysis. However, secondary CD34+ liquid cultures reselected from the primary liquid cultures 24 hours after HIV exposure by panning with the ICH3 CD34 MoAb (ICH3/CD34+) and maintained for an additional 14 days were negative for HIV expression. The ICH3-unbound cells were positive for both spliced and unspliced HIV RNA when exposed to HIV-1 Ba-L, and were DNA PCR positive when exposed to either monocytotropic or lymphotropic HIV-1. To further test that CD34+ cells were not infectible by HIV-1, we exposed K6.1/CD34+ cells continuously to HIV-1 in a culture system capable of maintaining and expanding primitive CD34+ cells. HIV-exposed K6.1/CD34+ cells proliferated and expanded as efficiently as uninfected cultures. However, when reselected magnetically using the K6.1 CD34 MoAb after expansion for 7 days, bound K6.1/CD34+ cells were again negative for HIV-1 expression, whereas unbound cells were positive for HIV-1 expression. These findings suggest that a sequential CD34+ cell-selection process, in which the two selections are separated by a brief culture period, can yield a population of CD34+ cells that are not infected with HIV-1. This process may be useful in the design of stem or progenitor cell-based transplantation therapies for HIV infection.

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.

Herpes simplex virus trans-regulatory protein ICP27 stabilizes and binds to 3' ends of labile mRNA.

Previous work demonstrated that a herpes simplex virus type 1 (HSV-1) immediate-early function up-regulates beta interferon but not chloramphenicol acetyltransferase reporter genes driven by the strong simian virus 40 (SV40) or cytomegalovirus promoter-enhancer regions in both transient assays and stable cell lines. The different 3' mRNA stabilization and RNA-processing signals from these two reporter genes appeared to be primarily responsible for this phenomenon. We now report that the HSV-1 ICP27 itself is sufficient to stimulate both steady-state accumulation and increased half-life of beta interferon reporter gene mRNA. Furthermore, the ability to respond directly to cotransfected ICP27 can be transferred to chloramphenicol acetyltransferase reporter genes by replacement of their SV40-derived splicing and poly(A) signals with the 3' AU-rich and poly(A) RNA-processing signals from the normally highly labile beta interferon and c-myc mRNA species. ICP27 expressed in bacteria bound specifically to in vitro-generated RNA from both the beta interferon and c-myc intronless AU-rich 3' RNA-processing regions, but not to the SV40-derived early-region splice signal and poly(A) sequences. By site-specific mutagenesis, we also show that individual ICP27 C-terminal amino acid residues that are positionally conserved in ICP27 homologs in other herpesviruses (D-357, E-358, H-479, C-400, C-483, and C-488) are critical for trans-regulatory activity. Importantly, several of these positions match mutations that are known to be essential for the role of ICP27 in the early-to-late switch during the virus lytic cycle. Therefore, our findings support the notion that HSV ICP27 modulates gene expression posttranscriptionally in part by targeting RNA.

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

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.

Evaluation of colocalization interactions between the IE110, IE175, and IE63 transactivator proteins of herpes simplex virus within subcellular punctate structures.

A number of previous studies have implied that three herpes simplex virus-encoded nuclear transactivator proteins, IE175 (ICP4), IE110 (ICP0), and IE63 (ICP27), may cooperate in transcriptional and posttranscriptional stimulation of viral gene expression. Using double-label immunofluorescence assays (IFA) in transient expression assays, we have examined the intracellular localization of these three proteins in DNA-transfected cells. The IE110 protein on its own forms spherical punctate domains within the nucleus, whereas the IE175 and IE63 proteins alone give uniform and speckled diffuse patterns, respectively. In infected cells, the IE110 punctate granules have been shown to correspond to novel preexisting subnuclear structures referred to as ND10 domains or PODs that contain a variety of cellular proteins, including SP100 and the PML proto-oncogene product. Cotransfection experiments with wild-type nuclear forms of both IE175 and IE110 provided direct evidence for partial redistribution of IE175 into the same punctate granules that contained IE110. Surprisingly, nuclear forms of IE110 were found to move a cytoplasmic form of IE175 into nuclear punctate structures, and a cytoplasmic form of IE110 was able to retain nuclear forms of IE175 in cytoplasmic punctate structures. Therefore, the punctate characteristic of IE110 appeared to both dominate the interactions and override the normal nuclear localization signals. The domains responsible for the interaction mapped to between codons 518 and 768 in 1E110 and to between codons 835 and 1029 in IE175. Importantly, a truncated nuclear form of the 1,298-amino-acid IE175 protein, which lacked the C-terminal domain beyond codon 834, was found to be excluded from the IE110 punctate granules. Cotransfection of nuclear or cytoplasmic IE110 with a truncated nuclear form of IE63 also led to partial redistribution of IE63 into either nuclear or cytoplasmic punctate granules containing IE110. Both the IE63-IE110 and IE175-IE110 colocalization interactions were demonstrated in Vero cells but not in 293 cells. Consequently, they differ from IE110 self-interactions, which correlate with in vitro dimerization and occur efficiently in both cell types. These interactions may help to explain the altered promoter target specificity and synergism observed when IE175 is cotransfected with IE110 in transactivation studies.

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.

A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector system utilizing stable HIV-1 packaging cell lines.

We have constructed stable human immunodeficiency virus (HIV) packaging cell lines that when transfected with an HIV-based retroviral vector produce packaged vectors capable of transducing susceptible CD4+ cells. This HIV-1-based retroviral vector system has the potential for providing targeted delivery and regulated expression of immunogens or antiviral agents in CD4+ cells.