Vincent F. La Russa

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

ABSTRACT 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.

Interleukin‐17A: A T‐Cell‐Derived Growth Factor for Murine and Human Mesenchymal Stem Cells

Interleukin‐17A (IL‐17A) is a proinflammatory cytokine expressed in activated T‐cells. It is required for microbial host defense and is a potent stimulator of granulopoiesis. In a dose‐dependent fashion, IL‐17A expanded human mesenchymal stem cells (MSCs) and induced the proliferation of mature stroma cells in bone marrow‐derived stroma cultures. Recombinant human interleukin‐17A (rhIL‐17A) nearly doubled colony‐forming unit‐fibroblast (CFU‐f) frequency and almost tripled the surface area covered by stroma. In a murine transplant model, in vivo murine (m)IL‐17A expression enhanced CFU‐f by 2.5‐fold. Enrichment of the graft with CD4+ T‐cell resulted in a 7.5‐fold increase in CFU‐f in normal C57BL/6, but only threefold in IL‐17Ra−/− mice on day 14 post‐transplant. In this transplant model, in vivo blockade of IL‐17A in C57BL/6 mice resembled the phenotype of IL‐17Ra−/− mice. Approximately half of the T‐cell‐mediated effect on MSC recovery following radiation‐conditioned transplantation was attributed to the IL‐17A/IL‐17Ra pathway. Pluripotent MSCs have the potential of regenerating various tissues, and mature stroma cells are critical elements of the hematopoietic microenvironment (HME). The HME is pivotal for formation and maintenance of functional blood cells. As a newly identified stroma cell growth factor, IL‐17A might have potential applications for novel treatment approaches involving MSCs, such as tissue graft engineering.

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.

IL-17 Mobilizes Peripheral Blood Stem Cells with Short- and Long-Term Repopulating Ability in Mice

Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin−/lowc-kit+Sca1+). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45–75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.

Imatinib (STI571) 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; Gleevec®, 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;q11) 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.

Marrow Stem Cells, Mesenchymal Progenitor Cells, and Stromal Progeny

During development, embryonic stem cells (ES) have been thought traditionally to give rise to organ-restricted stem cells. This process is believed to occur as a continuum through adult life since these stem cells also give rise to lineage committed progenitor cells, which retain a certain degree of multipotentiality in adult tissues. An example is the CFU-GEMM of the hematopoietic system which renews the formed elements of the blood. Tissue restricted stem cells and progenitor cells have been described for all adult tissues including bone marrow, liver, and brain. Quiescent stem cells that are retained in adult hematopoietic and non-hematopoietic tissues may be recruited from the quiescent state (G0) and mobilized when tissues are injured or depleted. They eventually form committed progenitor cells which differentiate to mature (terminal) cells that express genes that are functionally tissue specific. Differences exist between the hematopoietic and nonhematopoietic regenerative processes. Injury to or depletion of hematopoietic cells provokes replacement of blood or immune elements by newly generated cells derived from committed progenitor cells in the marrow microenvironment. In contrast, repair mechanisms associated with non-hematopoietic tissues may involve cells which appear mature, but which have retained some degree of proliferation and differentiation potential. Examples are hepatocytes, skin fibroblasts, endothelial cells, and smooth muscle cells. Previously, the differentiation potential of mesenchymal progenitor cells (MPC) or stromal progenitor cells in the bone marrow has been thought to be limited to the generation of hematopoietic supportive stromal cells of

Efficient c-kit Receptor-Targeted Gene Transfer to Primary Human CD34-Selected Hematopoietic Stem Cells

ABSTRACT We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit+ human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer.

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.

Hematopoietic Stem Cells

In the broadest sense, stem cells are primitive cells endowed with extensive capacities to develop and differentiate into functional and mature tissues or organs. A small, self-maintaining sub-population of stem cells will provide a continuous supply of functional cells throughout the entire lifespan of the organism. Therefore, a critical characteristic defining the ‘stem cell’ is the ability for unrestricted self-renewal throughout life of the individual organism. A large portion of the basic stem cell research has been conducted in the context of developing strategies to overcome cancer chemotherapy induced hematopoietic toxicity. Therefore, the term ‘stem cells’ is often perceived to be associated primarily with hematopoiesis. Given the fast turnover of short-lived blood cells, and morphologic evidence for maturation of functional cells from primitive precursor cells, the existence of pluripotent hematopoietic stem cells (HSC) has been postulated decades ago. However, it has become evident that the existence of stem cells may not only be exclusive to lymphohematopoietic tissue. In fact, there is now emerging strong evidence supporting the existence of primitive mesenchymal precursor cells, called stromal progenitor cells, that have the ability to differentiate into other functional structures such as connective tissue, neuronal tissue and muscle (1,2). There is further evidence for the persistence of an omnipotent or totipotent stem cell within the hematopoietic compartment, which possibly can also differentiate into other, non-hematopoietic tissues (2–4). The exact characterization and knowledge of the basic stem cell biology is required as a first step to develop strategies of manipulating these unique cells. Controlled manipulation and genetic modification of HSC could have tremendous potential on all aspects of human disease. In this review article, we provide a broad overview on the concept of stem cells. The focus of this review is on HSC, the definition and identification of precursor cells using established assays, applications and manipulation of precursor cells and current and future clinical applications.