Gerri Dooner

Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation.

Numerous animal studies have demonstrated that adult marrow‐derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that under certain conditions, radiation‐injured lung induced expression of pulmonary epithelial cell‐specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell‐impermeable membrane. Lung‐conditioned media had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell‐specific RNA‐filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung‐derived microvesicles and suggest a novel mechanism for marrow cell‐directed repair of injured tissue.

The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic stem cells

OBJECTIVE Hematopoietic stem cell homing and engraftment is dramatically altered by cytokine exposure. These studies address the molecular mechanisms responsible for the observed changes in transplantation biology. METHODS Primitive murine hematopoietic stem cells were isolated by fluorescence-activated cell sorting of lineage depleted (Lin(-)) cells exhibiting low staining of Hoechst 33342 and rhodamine 123 dyes or Lin(-) cells bearing Sca. Adhesion receptor expression was examined by immunofluorescence and reverse transcriptase polymerase chain reaction. In vitro adhesion assays were employed to define binding interactions between stem cells and stroma or extracellular matrix proteins. RESULTS Adhesion of Lin(-)Sca+ cells to Dexter stroma could be blocked by about 90% with antibodies to PECAM-1, alphaa(4), or beta(1), and partially blocked by antibodies to alpha(5), CD44, or L-selectin. By immunofluorescence, about 30% of purified Lin(-)Ho(lo)Rho(lo) cells expressed alpha(4), alpha(5), beta(1), and L-selectin, about 15% expressed alpha(L) and alpha(6), half expressed PECAM-1, and none expressed alpha(1) or alpha(2). After 48 hours in expansion cytokines, only 9% of the cells expressed alpha(4) and none expressed beta(1), whereas alpha(L) expression was fully restored, PECAM-1 and L-selectin partially restored, CD44 expression was newly induced, and adhesion to both fibronectin and laminin was reduced. Adhesion to purified collagen, fibronectin, or laminin enhanced expression of beta(1) integrins. CONCLUSION Expansion cytokines that move quiescent primitive hematopoietic stem cells into S phase markedly altered adhesion receptor expression and reduced their functional binding to extracellular matrix, which could reduce engraftment after transplant.

The stem cell continuum : Cell cycle, injury, and phenotype lability

Abstract:  The phenotype of the hematopoietic stem cell is intrinsically labile and impacted by cell cycle and the effects of tissue injury. In published studies we have shown that there are changes in short‐ and long‐term engraftment, progenitor numbers, gene expression, and differentiation potential with cytokine‐induced cell cycle transit. Critical points here are that these changes are reversible and not unidirectional weighing, heavily against a hierarchical model of stem cell regulation. Furthermore, a number of studies have now established that stem cells separated by lineage depletion and selection for Sca‐1 or c‐kit or low rhodamine and Hoechst staining are in fact a cycling population. Last, studies on Hoechst separated “cycling” stem cells indicates that the observed phenotype shifts relate to phase of cell cycle and are not due to in vitro exposure to cytokines. These data suggest a continuum model of stem cell regulation and further indicate that this model holds for in vivo situations. Observations that marrow cells can convert to various tissue cells under different injury conditions continue to be published despite a small, but influential, number of negative studies. Our studies and those of others indicate that conversions of marrow‐derived cells to different tissue cells, such as skeletal muscle and lung, is critically dependent upon multiple variables, the most important of which is the presence of tissue injury. Variables which affect conversion of marrow cells to nonhematopoietic cells after in vivo transplantation include the nature and timing of the injury; marrow mobilization; the marrow cell type infused; the timing of cell infusion and the number of cells infused; the cell cycle state of the marrow cells, and other functional alterations in the marrow cells the treatment of the host mouse separate from specific injury; the mode of cell delivery; and possibly the presence of microvesicles from injured tissue. At least some of the highlighted negative reports on stem cell plasticity appear to be due to a failure to address these variables. Recently, we have observed that irradiated lung releases microvesicles which can enter marrow cells and lead to the marrow cells expressing lung‐specific mRNA and protein. This could provide an underlying mechanism for many of the plasticity phenomena. Altogether, marrow appears to represent a highly flexible ever‐changing cell system with the capacity to respond to products of injured cells and top repair a broad range of tissues.

Microvesicle Induction of Prostate Specific Gene Expression in Normal Human Bone Marrow Cells

PURPOSE Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. MATERIALS AND METHODS We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 μM polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. RESULTS Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. CONCLUSIONS Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles.

Marrow cell genetic phenotype change induced by human lung cancer cells.

Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancer-derived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor recurrence, or metastases. They also suggest that the tissue environment may alter cancer cell gene expression.

The Stem Cell Continuum

Abstract: Hematopoietic stem cells have been felt to exist in a hierarchical structure with a relatively fixed phenotype at each stage of differentiation. Recent studies on the phenotype of the marrow hematopoietic stem cell indicate that it is not a fixed entity, but rather that it fluctuates and shows marked heterogeneity. Past studies have shown that stem cell engraftment characteristics, adhesion protein, and gene expression varies with the phase of the cell cycle. More recently, we demonstrated that progenitor numbers and differentiation potential also vary reversibly during one cytokine‐induced cell cycle transit. We have also shown high levels of conversion of marrow cells to skeletal muscle and lung cells, indicating a different level of plasticity. Recently, we demonstrated that homing to lung and conversion to lung cells in a mouse transplant model also fluctuates reversibly with cell cycle transit. This could be considered plasticity squared. These data indicate that marrow stem cells are regulated on a continuum related to the cell cycle both as to hematopoietic and to nonhematopoietic differentiation.

Conversion potential of marrow cells into lung cells fluctuates with cytokine-induced cell cycle

Green fluorescent protein (GFP)-labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung-specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit the cell cycle by exposure to interleukin-3 (IL-3), IL-6, IL-11, and Steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G(1)/S interface of the cell cycle have a three-fold increase in cells that assume a nonhematopoietic or pulmonary epithelial cell phenotype and that this increase is no longer seen in late S/G(2). These cells have been characterized as GFP(+) CD45(-) and GFP(+) cytokeratin(+). Thus, marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine-induced cell cycle transit. Previous studies have shown that the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse the cell cycle, leading to a continuum model of stem cell regulation. The present study indicates that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

Gene expression fluctuations in murine hematopoietic stem cells with cell cycle progression.

Evolving data suggest that marrow hematopoietic stem cells show reversible changes in homing, engraftment, and differentiation phenotype with cell cycle progression. Furthermore, marrow stem cells are a cycling population. Traditional concepts hold that the system is hierarchical, but the information on the lability of phenotype with cycle progression suggests a model in which stem cells are on a reversible continuum. Here we have investigated mRNA expression in murine lineage negative stem cell antigen‐1 positive stem cells of a variety of cell surface epitopes and transcription regulators associated with stem cell identity or regulation. At isolation these stem cells expressed almost all cell surface markers, and transcription factors studied, including receptors for G‐CSF, GM‐CSF, and IL‐7. When these stem cells were induced to transit cell cycle in vitro by exposure to interleukin‐3 (IL‐3), Il‐6, IL‐11, and steel factor some (CD34, CD45R c‐kit, Gata‐1, Gata‐2, Ikaros, and Fog) showed stable expression over time, despite previously documented alterations in phenotype, while others showed variation of expression between and within experiments. These latter included Sca‐1, Mac‐1, c‐fms, and c‐mpl. Tal‐1, endoglin, and CD4. These studies indicate that defined marrow stem cells express a wide variety of genes at isolation and with cytokine induced cell cycle transit show marked and reversible phenotype lability. Altogether, the phenotypic plasticity of gene expression for murine stem cells indicates a continuum model of stem cell regulation and extends the model to reversible expression with cell cycle transit of mRNA for cytokine receptors and stem cell markers. J. Cell. Physiol. 214: 786–795, 2008.

Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice.

Umbilical cord blood transplantation is considered an alternative to traditional bone marrow transplantation for patients who do not have matched sibling donors. In this study, we examined the effects of ex vivo treatment of human cord blood cells with cytokine mixtures and assessed the ability of treated cells to engraft in NOD‐scid mice. We incubated the cord blood with a four‐factor cytokine mixture of interleukin (IL)‐3, IL‐6, IL‐11 and stem cell factor, or with a two‐factor cytokine mixture of thrombopoietin and flt‐3. Incubation of cord blood for 48 h with either cytokine mixture did not affect progenitor cell number or proliferative potential as measured by the high proliferative potential (HPP) assay. Cytokine‐treated cord blood injected into irradiated NOD‐scid mice resulted in multilineage human engraftment. Overall, incubation with cytokines resulted in variable levels of engraftment with different cord blood samples. Incubation of cord blood with the four‐factor cytokine mixture resulted in increased survival of irradiated NOD‐scid recipients. These results demonstrate that short‐term ex vivo treatment of human progenitor cells gives variable results on in vivo multipotential capabilities.

Heterogeneity of Non-Cycling and Cycling Synchronized Murine Hematopoietic Stem/Progenitor Cells

Purified long‐term multilineage repopulating marrow stem cells have been considered to be homogenous, but functionally these cells are heterogeneous. Many investigators urge clonal studies to define stem cells but, if stem cells are truly heterogeneous, clonal studies can only define heterogeneity. We have determined the colony growth and differentiation of individual lineage negative, rhodamine low, Hoechst low (LRH) stem cells at various times in cytokine culture, corresponding to specific cell cycle stages. These highly purified and cycle synchronized (98% in S phase at 40 h of culture) stem cells were exposed to two cytokine cocktails for 0, 18, 32, or 40 h and clonal differentiation assessed 14 days later. Total heterogeneity as to gross colony morphology and differentiation stage was demonstrated. This heterogeneity showed patterns of differentiation at different cycle times. These data hearken to previous suggestions that stem cells might be similar to radioactive isotopes; decay rate of a population of radioisotopes being highly predictable, while the decay of individual nuclei is heterogeneous and unpredictable (Till et al., 1964 ). Marrow stem cells may be most adequately defined on a population basis; stem cells existing in a continuum of reversible change rather than a hierarchy. J. Cell. Physiol. 222:57–65, 2010.