Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.

To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues.

Imatinib (ST1571) provides only limited selectivity for CML cells and treatment might be complicated by silent BCR-ABL genes.

Very promising results have been obtained in clinical trials on chronic-phase chronic myeloid leukemia (CP-CML) patients treated with imatinib mesylate (IM; Gleevecr, STI571), a BCR-ABL tyrosine kinase inhibitor. However, we found that IM caused considerable inhibition of normal hematopoietic progenitor cells upon treating control bone marrow (BM) cultures. In vitro IM treatment gave a decrease in the yield and size of colonies from BM of untreated CP-CML patients that was only two to three times that from the normal samples. Moreover, about 30% of myeloid progenitors (CFU-GM) from CML BM still formed colonies in the presence of IM, most of which had BCR-ABL RNA. About half of these treated colonies also displayed methylation of the internal ABL Pa promoter, a CML-specific epigenetic alteration, which was used in this study as a marker for BCR-ABL translocation-containing cells. However, ~5-8% of the treated or the untreated CML BM-derived colonies had no detectable BCR-ABL RNA by two or three rounds of RT-PCR despite being positive for the internal standard RNA and displaying hallmarks of CML, either t(9;22)(q34;ql 1) or ABL Pa methylation. Our results indicate that IM is only partially specific for CML progenitor cells compared to normal hematopoietic progenitor cells and suggest that some CML cells may have a silent BCR-ABL oncogene that could interfere with therapy.

Suppression of clonogenic potential of human bone marrow mesenchymal stem cells by HIV type 1: putative role of HIV type 1 tat protein and inflammatory cytokines.

Bone marrow abnormalities are frequently observed in HIV-1-infected individuals. Infection of marrow mesenchymal stem cells (MSCs) may abrogate their growth properties and hematopoietic supportive functions. To delineate the cell type infected, and factors responsible for the deleterious effects, human bone marrow cells were exposed to HIV-1 in vitro. By week 4, the ability of MSCs to form colonies of purely fibroblasts (CFU-F) and mixed colonies of fibroblasts and adipocytes (CFU-FA) was suppressed by 23 +/- 5 and 55 +/- 7%, respectively. The p24 concentration in culture supernatants steadily declined from 170 ng/ml in the inoculum to 134 +/- 30, 35 +/- 15, 2.3 +/- 3, and <0.02 ng/ml at the end of week 1, 2, 3, and 4, respectively. However, even at week 4, coculturing with MT-4 lymphocytes for 1 week dramatically increased p24 levels. Polymerase chain reaction (PCR) amplification, using HIV-1-specific primers, and in situ hybridization with an HIV-1 cDNA probe demonstrated the presence of virus-specific nucleic acids within stromal colonies. Coimmunostaining with antibody to CD83 implicated the presence of HIV-1 within dendritic progenitor cells. Immunostaining with HIV-1 Tat antibody demonstrated the presence of Tat protein and reverse transcriptase (RT)-PCR assays showed increased (160-220%) mRNA levels for inflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 1beta [IL-1beta], IL-6, and macrophage inflammatory protein 1alpha [MIP-1alpha]). A concentration-dependent decrease in CFU-STROs was observed on incubation with either Tat protein (1-100 ng/ml) or with TNF-alpha or IL-1beta (0.025-25 ng/ml). These results suggest that HIV-1 infection of stromal cells may produce inhibitory factors that suppress the clonogenic potential of MSCs.

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases.

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.

Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins.

Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.

Neuronal stem cells biology and plasticity.

Recent studies have suggested that stem cells are able to cross primordial tissue barriers. Their ability to respond to unrelated microenvironmental signals strongly suggest that they have greater potential than previously imagined especially for their future clinical use for the regeneration of tissues or even perhaps organ systems. In particular there is an intriguing reciprocal relationship between the hematopoietic and neuronal stem cell systems. Both stem cell pools appear to respond to microenvironmental signals that are not developmental related. These intriguing observations have led to a number of studies that have attempted to explore this apparent phenomenon of plasticity associated with both hematopoietic and neuronal stem cell populations.

Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells.

Bone marrow-derived mesenchymal stromal cells (MSCs) have attracted attention as potential platforms for the systemic delivery of therapeutic proteins in vivo following gene transfer using oncogenic retroviruses. However, the major limitations of this strategy include low levels of gene transfer and a general lack of long-term transgene expression. We have investigated the expression of several transgenes in MSCs following HIV-1 lentiviral vector-mediated gene transfer. Vectors containing a variety of strong promoters driving enhanced green fluorescence protein (EGFP) and coral (Discosoma sp.)-derived red fluorescent protein (DsRed) reporter genes pseudotyped with the vesicular stomatitis virus-G (VSV-G) glycoprotein were able to transduce cultured MSCs with high efficiency. Transduction efficiencies and transgene expression levels in MSCs were found to be higher with lentiviral vectors than with a vector based on the murine stem cell virus pseudotyped with VSV-G. Transgene expression was maintained in culture for at least 5 months. HIV-1-based lentiviral vectors were able to transduce clonogenic mesenchymal progenitor cells, which were capable of maintaining transgene expression by their MSC progeny, over several cell divisions and during differentiation into adipocytes, indicating that terminal adipocyte cell differentiation was unaffected by lentivirus-mediated reporter gene transfer. Collectively these results suggest that lentivirus-mediated gene transfer strategies provide an efficient tool for ex vivo modification of MSCs that does not interfere with differentiation.