Kevin W. Johnson

Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription

OBJECTIVE Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA. MATERIALS AND METHODS Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-microm pore size), were analyzed using species-specific primers (for rat or mouse). RESULTS These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena. CONCLUSION These studies suggest that cellular systems are more phenotypically labile than previously considered.

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 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.

Engraftment of retroviral EGFP-transduced bone marrow in mice prevents rejection of EGFP-transgenic skin grafts.

We have investigated whether a state of tolerance toward EGFP-expressing skin tissue can be induced by prior establishment of EGFP molecular chimerism by transplant of gene-transduced bone marrow in mice. Irradiated (10 Gy) C57BL/6J mice were transplanted with bone marrow cells transduced with two different retroviral vectors encoding EGFP. EGFP-transduced, mock-transduced, and age-matched control mice received skin grafts from both C57BL/6 EGFP-transgenic (B6-EGFP. Tg) and MHC-mismatched B10.A donor mice at 8, 29, or 39 weeks after bone marrow transplantation. Although 14 of 17 control mice rejected EGFP.Tg skin grafts within 100 days, 24 of 25 mice receiving EGFP-expressing bone marrow cells accepted their B6-EGFP.Tg grafts out to 200 days after skin grafting, including animals with undetectable levels of EGFP expression in blood cells. The EGFP-transduced animals rejected third-party grafts from MHC-mismatched mice within 20 days, indicating that acceptance of the EGFP-expressing skin grafts was the result of the induction of specific and operational immune tolerance. Thus, our data indicate that (a) EGFP-expressing tissue elicits an immunological rejection in C57BL/6 mice and (b) tolerance can be induced by engrafting relatively small numbers of EGFP-transduced hematopoietic cells. These experiments utilizing EGFP as an immunogen point to the wider therapeutic potential of employing transplantation of gene-transduced hematopoietic cells for establishing immunological tolerance and thereby preventing rejection of gene-corrected cells and tissues.

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.

The murine long-term multi-lineage renewal marrow stem cell is a cycling cell

Prevailing wisdom holds that hematopoietic stem cells (HSCs) are predominantly quiescent. Although HSC cycle status has long been the subject of scrutiny, virtually all marrow stem cell research has been based on studies of highly purified HSCs. Here we explored the cell cycle status of marrow stem cells in un-separated whole bone marrow (WBM). We show that a large number of long-term multi-lineage engraftable stem cells within WBM are in S/G2/M phase. Using bromodeoxyuridine, we show rapid transit through the cell cycle of a previously defined relatively dormant purified stem cell, the long-term HSC (LT-HSC; Lineage−/c-kit+/Sca-1+/Flk-2−). Actively cycling marrow stem cells have continually changing phenotype with cell cycle transit, likely rendering them difficult to purify to homogeneity. Indeed, as WBM contains actively cycling stem cells, and highly purified stem cells engraft predominantly while quiescent, it follows that the population of cycling marrow stem cells within WBM are lost during purification. Our studies indicate that both the discarded lineage-positive and lineage-negative marrow cells in a stem cell separation contain cycling stem cells. We propose that future work should encompass this larger population of cycling stem cells that is poorly represented in current studies solely focused on purified stem cell populations.

Homing and Long-Term Engraftment of Long- and Short- Term Renewal Hematopoietic Stem Cells

Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.