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Mesenchymal stem cell extracellular vesicles attenuate pulmonary hypertension, but their ability to reverse established disease in larger animal models and the duration and mechanism(s) of their effect are unknown. We sought to determine the efficacy and mechanism of mesenchymal stem cells’ extracellular vesicles in attenuating pulmonary hypertension in rats with Sugen/hypoxia-induced pulmonary hypertension. Male rats were treated with mesenchymal stem cell extracellular vesicles or an equal volume of saline vehicle by tail vein injection before or after subcutaneous injection of Sugen 5416 and exposure to 3 weeks of hypoxia. Pulmonary hypertension was assessed by right ventricular systolic pressure, right ventricular weight to left ventricle + septum weight, and muscularization of peripheral pulmonary vessels. Immunohistochemistry was used to measure macrophage activation state and recruitment to lung. Mesenchymal stem cell extracellular vesicles injected before or after induction of pulmonary hypertension normalized right ventricular pressure and reduced right ventricular hypertrophy and muscularization of peripheral pulmonary vessels. The effect was consistent over a range of doses and dosing intervals and was associated with lower numbers of lung macrophages, a higher ratio of alternatively to classically activated macrophages (M2/M1 = 2.00 ± 0.14 vs. 1.09 ± 0.11; P

The role of bone marrow (BM) cells in modulating pulmonary hypertensive responses is not well understood. Determine if BM-derived endothelial progenitor cells (EPCs) induce pulmonary hypertension (PH) and if this is attenuated by mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). Three BM populations were studied: (a) BM from vehicle and monocrotaline (MCT)-treated mice (PH induction), (b) BM from vehicle-, MCT-treated mice that received MSC-EV infusion after vehicle, MCT treatment (PH reversal, in vivo), (c) BM from vehicle-, MCT-treated mice cultured with MSC-EVs (PH reversal, in vitro). BM was separated into EPCs (sca-1+/c-kit+/VEGFR2+) and non-EPCs (sca-1-/c-kit-/VEGFR2-) and transplanted into healthy mice. Right ventricular (RV) hypertrophy was assessed by RV-to-left ventricle+septum (RV/LV+S) ratio and pulmonary vascular remodeling by blood vessel wall thickness-to-diameter (WT/D) ratio. EPCs but not non-EPCs from mice with MCT-induced PH (MCT-PH) increased RV/LV+S, WT/D ratios in healthy mice (PH induction). EPCs from MCT-PH mice treated with MSC-EVs did not increase RV/LV+S, WT/D ratios in healthy mice (PH reversal, in vivo). Similarly, EPCs from MCT-PH mice treated with MSC-EVs pre-transplantation did not increase RV/LV+S, WT/D ratios in healthy mice (PH reversal, in vitro). MSC-EV infusion reversed increases in BM-EPCs and increased lung tissue expression of EPC genes and their receptors/ligands in MCT-PH mice. These findings suggest that the pulmonary hypertensive effects of BM are mediated by EPCs and those MSC-EVs attenuate these effects. These findings provide new insights into the pathogenesis of PH and offer a potential target for development of novel PH therapies.

Mesenchymal stromal cells (MSCs) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 h to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 h post irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% dimethyl sulfoxide at -80 °C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling and ultimately death. Two rodent models of PH include treatment with monocrotaline or exposure to a vascular endothelial growth factor receptor inhibitor and hypoxia. Studies in these models indicated that damaged lung cells evolve extracellular vesicles which induce production of progenitors that travel back to the lung and induce PH. A study in patients with pulmonary myelofibrosis and PH indicated that 100 cGy lung irradiation could remit both diseases. Previous studies indicated that murine progenitors were radiosensitive at very low doses, suggesting that 100 cGy treatment of mice with induced PH might be an effective PH therapy. Our hypothesis is that the elimination of the PH‐inducing marrow cells by low dose irradiation would remove the cellular influences creating PH. Here we show that low dose whole‐body irradiation can both prevent and reverse established PH in both rodent models of PH.

Extracellular vesicles (EVs) are important mediators of intercellular communication and have been implicated in myriad physiologic and pathologic processes within the hematopoietic system. Numerous factors influence the ability of EVs to communicate with target marrow cells, but little is known about how circadian oscillations alter EV function. In order to explore the effects of daily rhythms on EV-mediated intercellular communication, we used a well-established model of lung-derived EV modulation of the marrow cell transcriptome. In this model, co-culture of whole bone marrow cells (WBM) with lung-derived EVs induces expression of pulmonary specific mRNAs in the target WBM. To determine if daily rhythms play a role in this phenotype modulation, C57BL/6 mice were entrained in 12-hour light/12-hour dark boxes. Lungs harvested at discrete time-points throughout the 24-hour cycle were co-cultured across a cell-impermeable membrane with murine WBM. Alternatively, WBM harvested at discrete time-points was co-cultured with lung-derived EVs. Target WBM was collected 24hrs after co-culture and analyzed for the presence of pulmonary specific mRNA levels by RT-PCR. In both cases, there were clear time-dependent variations in the patterns of pulmonary specific mRNA levels when either the daily time-point of the lung donor or the daily time-point of the recipient marrow cells was altered. In general, WBM had peak pulmonary-specific mRNA levels when exposed to lung harvested at Zeitgeber time (ZT) 4 and ZT 16 (ZT 0 defined as the time of lights on, ZT 12 defined as the time of lights off), and was most susceptible to lung-derived EV modulation when target marrow itself was harvested at ZT 8- ZT 12. We found increased uptake of EVs when the time-point of the receptor WBM was between ZT 20 -ZT 24, suggesting that the time of day-dependent changes in transcriptome modulation by the EVs were not due simply to differential EV uptake. Based on these data, we conclude that circadian rhythms can modulate EV-mediated intercellular communication.

Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently, vesicles were studied as possible vaccines and, most recently, there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either the lung or liver can enter bone marrow cells (this is a prerequisite) and alter their fate toward that of the originating liver and lung tissue. Lung vesicles interacted with bone marrow cells result in the bone marrow cells expressing surfactants A–D, Clara cell protein, and aquaporin-5 mRNA. In a similar vein, liver-derived vesicles induce albumin mRNA in target marrow cells. The vesicles contain protein, mRNA, microRNA, and noncoding RNA and variably some DNA. This genetic package is delivered to cells and alters the phenotype. Further studies have shown that initially the altered phenotype is due to the transfer of mRNA and a transcriptional modulator, but long-term epigenetic changes are induced through transfer of a transcriptional factor, and the mRNA is rapidly degraded in the cell. Studies on the capacity of vesicles to restore injured tissue have been quite informative. Mesenchymal stem cell-derived vesicles are able to reverse the injury to the damaged liver and kidney. Other studies have shown that mesenchymal stem cell-derived vesicles can reverse radiation toxicity of bone marrow stem cells. Extracellular vesicles offer an intriguing strategy for treating a number of diseases characterized by tissue injury.

We have shown that hematopoietic stem/progenitor cell phenotype and differentiative potential change throughout cell cycle. Lung-derived microvesicles (LDMVs) also change marrow cell phenotype by inducing them to express pulmonary epithelial cell-specific mRNA and protein. These changes are accentuated when microvesicles isolated from injured lung. We wish to determine if microvesicle-treated stem/progenitor cell phenotype is linked to cell cycle and to the injury status of the lung providing microvesicles. Lineage depleted, Sca-1+ (Lin-/Sca-1+) marrow isolated from mice were cultured with interleukin 3 (IL-3), IL-6, IL-11, and stem cell factor (cytokine-cultured cells), removed at hours zero (cell cycle phase G0/G1), 24 (late G1/early S), and 48 (late S/early G2/M), and cocultured with lung tissue, lung conditioned media (LCM), or LDMV from irradiated or nonirradiated mice. Alternatively, Lin-/Sca-1+ cells not exposed to exogenous cytokines were separated into G0/G1 and S/G2/M cell cycle phase populations by fluorescence-activated cell sorting (FACS) and used in coculture. Separately, LDMV from irradiated and nonirradiated mice were analyzed for the presence of adhesion proteins. Peak pulmonary epithelial cell-specific mRNA expression was seen in G0/G1 cytokine-cultured cells cocultured with irradiated lung and in late G1/early S cells cocultured with nonirradiated lung. The same pattern was seen in cytokine-cultured Lin-/Sca-1 cells cocultured with LCM and LDMV and when FACS-separated Lin-/Sca-1 cells unexposed to exogenous cytokines were used in coculture. Cells and LDMV expressed adhesion proteins whose levels differed based on cycle status (cells) or radiation injury (LDMV), suggesting a mechanism for microvesicle entry. These data demonstrate that microvesicle modification of progenitor/stem cells is influenced by cell cycle and the treatment of the originator lung tissue.

There have been reports of marrow cells converting into pulmonary epithelial cells after marrow transplantation in irradiated mice. We evaluated the impact of whole bone marrow (WBM) infusion in mice, with or without total body irradiation (TBI), treated with saline or monocrotaline (MCT), which induces pulmonary hypertension (PH). C57BL/6 mice were injected with MCT or saline weekly for 4 weeks. Cohorts were then infused with saline vehicle (vehicle) or WBM from C57BL/-Tg(UBC-GFP)30Scha/J mice, with or without previous TBI (WBM or WBM/TBI). Four weeks later, right ventricular peak pressures (RVPP), right ventricular free wall-to-body weight ratios (RV/BW), and pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were determined. WBM infusion and WBM following TBI induced increases in RVPP and RV/BW in saline-treated mice, while only TBI-exposed mice showed additional increases in PVWT/D. MCT increased RVPP, RV/BW, and PVWT/D in mice given vehicle or WBM alone, but not in mice given WBM/TBI. RVPP and RV/BW were not significantly lower in MCT mice given WBM/TBI than in MCT mice treated with vehicle, but MCT-treated mice given WBM or TBI/WBM had significantly lower PVWT/D compared to MCT-treated mice given saline vehicle. No donor WBM-derived pulmonary vascular cells were detected, suggesting that the observed effects of WBM infusion may be due to paracrine effects separate from cell conversions. The observation of PH after marrow infusion suggests an additional mechanism for lung toxicity seen in marrow transplantation. In conclusion, WBM alone appears to increase RVPP and RV/BW in normal mice but the combination of WBM and TBI attenuates MCT-induced PH.

Cell-based therapy in adult lung injury models is associated with highly variable donor cell engraftment and epithelial reconstitution. The role of marrow-derived cell therapy in neonatal lung injury is largely unknown. In this study, we determined the fate and effects of adult bone marrow cells in a model of neonatal lung injury. Wild-type mice placed in a normoxic or hyperoxic (95% O(2)) environment received bone marrow cells from animals expressing green fluorescent protein (GFP) at Postnatal Day (P)5. Controls received vehicle buffer. Lungs were analyzed between Post-Transplantation (TPX) Day 2 and Week 8. The volume of GFP-immunoreactive donor cells, monitored by stereologic volumetry, remained constant between Post-TPX Weeks 1 and 8 and was similar in normoxic and hyperoxia-exposed recipients. Virtually all marrow-derived cells showed colocalization of GFP and the pan-macrophage marker, F4/80, by double immunofluorescence studies. Epithelial transdifferentiation was not seen. Marrow cell administration had adverse effects on somatic growth and alveolarization in normoxic mice, while no effects were discerned in hyperoxia-exposed recipients. Reexposure of marrow-treated animals to hyperoxia at P66 resulted in significant expansion of the donor-derived macrophage population. In conclusion, intranasal administration of unfractionated bone marrow cells to newborn mice does not achieve epithelial reconstitution, but establishes persistent alveolar macrophage chimerism. The predominantly adverse effects of marrow treatment in newborn lungs are likely due to macrophage-associated paracrine effects. While this model and route of cell therapy may not achieve epithelial reconstitution, the role of selected stem cell populations and/or alternate routes of administration for cell-based therapy in injured newborn lungs deserve further investigation.

Marrow stem cell regulation represents a complex and flexible system. It has been assumed that the system was intrinsically hierarchical in nature, but recent data has indicated that at the progenitor/stem cell level the system may represent a continuum with reversible alterations in phenotype occurring as the stem cells transit cell cycle. Short and long-term engraftment, in vivo and in vitro differentiation, gene expression, and progenitor numbers have all been found to vary reversibly with cell cycle. In essence, the stem cells appear to show variable potential, probably based on transcription factor access, as they proceed through cell cycle. Another critical component of the stem cell regulation is the microenvironment, so-called niches. We propose that there are not just several unique niche cells, but a wide variety of niche cells which continually change phenotype to appropriately interact with the continuum of stem cell phenotypes. A third component of the regulatory system is microvesicle transfer of genetic information between cells. We have shown that marrow cells can express the genetic phenotype of pulmonary epithelial cells after microvesicle transfer from lung to marrow cells. Similar transfers of tissue specific mRNA occur between liver, brain, and heart to marrow cells. Thus, there would appear to be a continuous genetic modulation of cells through microvesicle transfer between cells. We propose that there is an interactive triangulated Venn diagram with continuously changing stem cells interacting with continuously changing areas of influence, both being modulated by transfer of genetic information by microvesicles.

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.Disclosure of potential conflicts of interest is found at the end of this article.

Background : Our group has previously demonstrated that murine whole bone marrow cells (WBM) that internalize lung-derived extracellular vesicles (LDEVs) in culture express pulmonary epithelial cell–specific genes for up to 12 weeks. In addition, the lungs of lethally irradiated mice transplanted with lung vesicle–modulated marrow have 5 times more WBM-derived type II pneumocytes compared to mice transplanted with unmanipulated WBM. These findings indicate that extracellular vesicle modification may be an important consideration in the development of marrow cell–based cellular therapies. Current studies were performed to determine the specific marrow cell types that LDEV stably modify. Methods : Murine WBM-derived stem/progenitor cells (Lin-/Sca-1+) and differentiated erythroid cells (Ter119+), granulocytes (Gr-1+) and B cells (CD19+) were cultured with carboxyfluorescein N -succinimidyl ester (CFSE)-labelled LDEV. LDEV+ cells (CFSE+) and LDEV− cells (CFSE−) were separated by flow cytometry and visualized by fluorescence microscopy, analyzed by RT-PCR or placed into long-term secondary culture. In addition, murine Lin-/Sca-1+ cells were cultured with CFSE-labelled LDEV isolated from rats, and RT-PCR analysis was performed on LDEV+ and–cells using species-specific primers for surfactant (rat/mouse hybrid co-cultures). Results : Stem/progenitor cells and all of the differentiated cell types studied internalized LDEV in culture, but heterogeneously. Expression of a panel of pulmonary epithelial cell genes was higher in LDEV+cells compared to LDEV− cells and elevated expression of these genes persisted in long-term culture. Rat/mouse hybrid co-cultures revealed only mouse-specific surfactant B and C expression in LDEV+ Lin-/Sca-1+cells after 4 weeks of culture, indicating stable de novo gene expression. Conclusions : LDEV can be internalized by differentiated and more primitive cells residing in the bone marrow in culture and can induce stable de novo pulmonary epithelial cell gene expression in these cells for several weeks after internalization. The gene expression represents a transcriptional activation of the target marrow cells. These studies serve as the basis for determining marrow cell types that can be used for cell-based therapies for processes that injure the pulmonary epithelial surfaces. Keywords: bone marrow cells; pulmonary epithelial cells; extracellular vesicles (Published: 16 September 2015) Citation: Journal of Extracellular Vesicles 2015, 4: 26166 - http://dx.doi.org/10.3402/jev.v4.26166

Growing evidence suggests that transcriptional regulators and secreted RNA molecules encapsulated within membrane vesicles modify the phenotype of target cells. Membrane vesicles, actively released by cells, represent a mechanism of intercellular communication that is conserved evolutionarily and involves the transfer of molecules able to induce epigenetic changes in recipient cells. Extracellular vesicles, which include exosomes and microvesicles, carry proteins, bioactive lipids, and nucleic acids, which are protected from enzyme degradation. These vesicles can transfer signals capable of altering cell function and/or reprogramming targeted cells. In the present review we focus on the extracellular vesicle-induced epigenetic changes in recipient cells that may lead to phenotypic and functional modifications. The relevance of these phenomena in stem cell biology and tissue repair is discussed.

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.

Adult stem cells are likely to have much more versatile differentiation capabilities than once believed. Numerous studies have appeared over the past decade demonstrating the ability of adult stem cells to differentiate into a variety of cells from non-hematopoietic organs, including the lung. The goal of this review is to provide an overview of the growth factors which are thought to be involved in lung development and disease, describe the cells within the lung that are believed to replace cells that have been injured, review the studies that have demonstrated the transformation of bone marrow-derived stem cells into lung cells, and describe potential clinical applications with respect to human pulmonary disease.

The conception of the present-day model of hematopoiesis was begun by the work of Professor Ernst Neumann in the 19th century when he established that immature blood cells in the bone marrow migrate out into the blood vessels. Here was the birth of the hierarchical model of hematopoiesis. Jumping 135 years into the present day, recent data suggests that the stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum. Presumptively, chromatin remodeling with cycle transit underlies changes in gene expression. This implies that the differentiative potential of primitive stem cells should also shift with cycle transit. This model proposes a less rigid system, at least in the early stem cell and progenitor compartments in which the functional characteristics of stem cells change as they go through cycle transit. We have shown that hematopoietic stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Other shifts include adhesion protein expression, cytokine receptor expression, gene expression, and progenitor phenotype. We have also found differentiation “hotspots”, culture times (reflective of cell cycle state) at which stem cell differentiation was directed toward a specific lineage. This data inaugurates the end of a pure stochastic model. This work complements existing scientific work without discounting it and adds an additional dimension of complexity (or simplicity) to the process of hematopoiesis.

Current concepts of hematopoiesis are encompassed in a hierarchical stem cell model. This developed initially from studies of colony-forming unit spleen and in vitro progenitors for different cell lineages, but then evolved into a comprehensive model of cells with different in vivo differentiative and proliferative potential. These cells were characterized and purified based largely on expression of a variety of lineage-specific and stem cell-specific surface epitopes. Monoclonal antibodies were bound to these epitopes and then used to physically and fluorescently separate different classes of these cells. The gold standard for the most primitive marrow stem cells was long-term multilineage repopulation and renewal in lethally irradiated mice. Progressive work seemed to have clonally defined a Lineage negative (Lin−), Sca-1+, c-kit+, CD150+ stem cell with great proliferative, differentiative, and renewal potential. This cell was stable and in the G0 phase of cell cycle. However, continued work in our laboratory indicated that the engraftment, differentiation, homing, and gene expression phenotype of the murine marrow stem cells continuously and reversibly changes with passage through cell cycle. Most recently, using cycle-defining supravital dyes and fluorescent-activated cell sorting and S-phase-specific tritiated thymidine suicide, we have established that the long-term repopulating hematopoietic stem cell is a rapidly proliferating, and thus a continually changing cell; as a corollary it cannot be purified or defined on a clonal single cell basis. Further in vivo studies employing injected and ingested 5-Bromodeoxyuridine (BrdU), showed that the G0 Lin-Sca-1, c-kit+ Flt3− cell was rapidly passing through cell cycle. These data are explained by considering the separative process: the proliferating stem cells are eliminated through the selective separations leaving non-representative dormant G0 stem cells. In other words, they throw out the real stem cells with the purification. This system, where the marrow stem cell continuously and reversibly changes with obligate cell cycle transit, is further complicated by the consideration of the impact of tissue microvesicles on the cell phenotypes. Tissue microvesicles have been found to alter the phenotype of marrow cells, possibly explaining the observations of “stem cell plasticity.” These alterations, short-term, are due to transfer of originator cell mRNA and as yet undefined transcription factors. Long-term phenotype change is due to transcriptional modulation; a stable epigenetic change. Thus, the stem cell system is characterized by continuous cycle and microvesicle-related change. The challenge of the future is to define the stem cell population.

Marrow cells are a rich source of vesicles (released from their surface or endosomal compartment that may deliver proteins, ribonucleic acid (RNA), and microRNA (miRNA) into damaged organs. Based on this, paracrine effects probably make major contributions in most of the currently reported positive results in clinical trials employing adult hematopoietic stem cells. In this chapter, we will discuss our data showing that extracellular vesicles might have significant therapeutic potential in various tissue injuries as well as in treatment of cancer.

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.

Background: Interest has been generated in the capacity of cellular-derived microvesicles to alter the fate of different target cells. Lung, liver, heart and brain-derived vesicles can alter the genetic phenotype of murine marrow cells; however, the stability of such changes and the mechanism of these changes remain unclear. In the present work, we show that lung-derived microvesicles (LDMV) alter the transcriptome and proteome of target marrow cells initially by mRNA and regulator(s) of transcription transfer, but that long term phenotype change is due solely to transfer of a transcriptional regulator with target cell. Methods/results: In vivo studies: Whole bone marrow cells (WBM) were co-cultured with LDMV (both isolated from male C57BL/6 mice) or cultured alone (control). One week later, cultured WBM was transplanted into lethally-irradiated female C57BL/6 mice. Recipient mice were sacrificed 6 weeks later and WBM, spleens and livers were examined for the presence of lung-specific gene expression, including surfactants A, B, C and D, aquaporin-5, and clara cell specific protein, via real-time RT-PCR. Immunohistochemistry was also performed on lungs to determine the number of transplanted marrowderived (Y chromosome+) type II pneumocytes (prosurfactant C+). Mice transplanted with LDMV cocultured WBM expressed pulmonary epithelial cell genes in the cells of their bone marrow, livers and spleens and over fivefold more transplanted marrow-derived Y+/prosurfactant C+cells could be found in their lungs (vs. control mice). In vitro studies: WBM (from mice or rats) was cultured with or without LDMV (from mice or rats) for 1 week then washed and cultured alone. WBM was harvested at 2-week intervals for real-time RT-PCR analysis, using species-specific surfactant primers, and for Western Blot analysis. Proteomic and microRNA microarray analyses were also performed on cells. LDMV co-cultured WBM maintained expression of pulmonary epithelial cell genes and proteins for up to 12 weeks in culture. Surfactant produced at later time points was specific only to the species of the marrow cell in culture indicating de novo mRNA transcription. These findings, in addition to the altered protein and microRNA profiles of LDMV co-cultured WBM, support a stable transcriptional mechanism for these changes. Conclusions: These data indicate that microvesicle alteration of cell fate is robust and long-term and represents an important new aspect of cellular biology. Keywords: bone marrow cells; lung; microvesicles; bone marrow transplant; transcription factor (Published: 16 April 2012) Citation: Journal of Extracellular Vesicles 2012, 1 : 18163 - http://dx.doi.org/10.3402/jev.v1i0.18163

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.

Cell phenotype alteration by cell-derived vesicles presents a new aspect for consideration of cell fate. Accumulating data indicates that vesicles from many cells interact with or enter different target cells from other tissues, altering their phenotype toward that of the cell releasing the vesicles. Cells may be changed by direct interactions, transfer of cell surface receptors or epigenetic reprogramming via transcriptional regulators. Induced epigenetic changes appear to be stable and result in significant functional effects. These data force a reconsideration of the cellular context in which transcription regulates the proliferative and differentiative fate of tissues and suggests a highly plastic cellular system, which might underlay a relatively stable tissue system. The capacity of marrow to convert to non-hematopoietic cells related to vesicle cross-communication may underlie the phenomena of stem cell plasticity. Additionally, vesicles have promise in the clinical arenas of disease biomarkers, tissue restoration and control of neoplastic cell growth.

The phenotype of marrow hematopoietic stem cells is determined by cell cycle state and microvesicle entry into the stem cells. The stem cell population is continually changing based on cell cycle transit and thus can only be defined on a population basis. Purification of marrow stem cells only addresses the heterogeneity of these populations. When whole marrow is studied, the long-term repopulating stem cells are in active cell cycle. However, with some variability, when highly purified stem cells are studied, the cells appear to be dormant. Thus, the study of purified stem cells is intrinsically misleading. Tissue-derived microvesicles enhanced by injury effect the phenotype of different cell classes. We propose that previously described stem cell plasticity is due to microvesicle modulation. We further propose a stem cell population model in which the individual cell phenotypes continually changes, but the population phenotype is relatively stable. This, in turn, is modulated by microvesicle and microenvironmental influences.

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-mu m pore sire), were analyzed using species-specific printers (for rat or mouse). Results. These studies revealed that surfactant 13 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. (C) 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

We tracked the extracellular fate of proteins of pulmonary origin using the technique of stable isotope labeling of amino acids in cell culture (SILAC) in cell-impermeable Transwell culture systems. We find that irradiation to murine lung and lung-derived cells induces their release of proteins that are capable of entering neighboring cells, including primary murine bone marrow cells as well as prostate cancer and hematopoietic cell lines. The functional classification of transferred proteins was broad and included transcription factors, mediators of basic cellular processes and components of the nucleosome remodeling and deacetylase complex, including metastasis associated protein 3 and retinoblastoma-binding protein 7. In further analysis we find that retinoblastoma-binding protein 7 is a transcriptional activator of E-cadherin and that its intercellular transfer leads to decreased gene expression of downstream targets such as N-cadherin and vimentin. SILAC-generated data sets offer a valuable tool to identify and validate potential paracrine networks that may impact relevant biologic processes associated with phenotypic and genotypic signatures of health and disease.

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.

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 cell cycle by exposure to 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 G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. 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 the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

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.

Rationale: We have demonstrated that whole, undifferentiated bone marrow (WBM) cells isolated from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) cause pulmonary hypertensive changes when transplanted into lethally-irradiated mice (Aliotta et al., 2014 ATS abstract). In addition, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs), sub-cellular particles shed by activated cells, can reverse features of MCT-induced PH (Aliotta et al., 2015 ATS abstract). Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis of human PH as more have been noted to be present in the circulation and remodeled pulmonary vasculature of these patients compared with healthy humans. We wished to determine if bone marrow-derived EPCs isolated from mice with MCT PH were the cells responsible for inducing PH in healthy mice and if so, whether exposure of these EPCs to MSC EVs prevented the development of PH. Methods: Sca-1+/ckit+/flk1+ cells (EPCs) and sca-1-/ckit-/flk1- cells (non-EPCs) were isolated from the bone marrow of MCT-injured and vehicle-injected mice. Cohorts of lethally-irradiated mice were transplanted with EPCs (2,000 EPCs + 300,000 helper WBM cells/recipient) or non-EPCs (200,000 non-EPCs + 200,000 helper WBM cells/recipient) from the bone marrow of MCT-injured and vehicle-injected mice. 28 days after transplantation, recipient mouse right ventricular (RV) hypertrophy was assessed by RV-to-left ventricle+septum (RV/LV+S) ratio (mg/g) and pulmonary vascular remodeling by blood vessel wall thickness-to-diameter (WT/D) ratio. In addition, WBM from MCT-injured and vehicle-injected mice was cultured with or without MSC EVs for 48 hours. EPCs (80-100 EPCs + 300,000 helper WBM cells/recipient) and non-EPCs (500,000-700,000 non- EPCs + 300,000 helper WBM cells/recipient) were separated by flow cytometry and infused into lethally-irradiated mice which were analyzed 28 days post-transplant. Results: RV/LV+S ratios of mice transplanted with EPCs from MCT-injured mice (0.256+/-0.142 mg/g) were similar to those of MCT-injured mice (0.274+/-0.211 mg/g, p=NS, n=5/cohort) but elevated compared to mice transplanted with EPCs and non-EPCs from vehicle-injected mice (0.126+/-0.111 and 0.121+/-0.134 mg/g, p Conclusions: These findings suggest that EPCs are among the pathogenic bone marrow cells in MCT-injured mice that are responsible for inducing RV hypertrophy and pulmonary vascular remodeling, key features of PH, upon transplantation into healthy mice. MSC EVs appear to be capable of reversing this disease via interactions with EPCs. Disclosures No relevant conflicts of interest to declare.

We have shown that Human or mouse mesenchymal stem cell (MSC) derived vesicles separated by differential centrifugation (300g, 10kg &100kg) can reverse radiation whole bone marrow (WBM) damage in vitro on the hematopoietic cell line FDC-P1 and in vivo on recovery of stem cells and differentiated blood cells after whole body irradiation. The vesicle separation is a classic method for preparations of exosomes. However, this separation isolates the smaller vesicles, discarding larger vesicles. Biologic healing effects of larger vesicles with or without smaller vesicles have not been investigated. Here, we investigated the healing effects of 3 different preparations of vesicles: 10k pellet (microvesicles), 100-10k pellet (exosomes) & 100k pellet (no 10k spin). Three different fractions (10k pellet, 100-10k pellet &100k pellet) of murine/human MSC or murine WBM derived vesicles were isolated by differential centrifugation. FDC-P1 cells were exposed at 500cGy irradiation and cultured with or without the addition of 3 different preparations of murine WBM or human MSC-derived vesicles for 7 days. A significant increase in cell proliferation was observed after 3 different vesicle fractions treatments. However, exosomes were clearly inferior, microvesicles and the combined exosomes and microvesicles population were superior. The capacity of human MSC-derived vesicles of different fractions on reversal of murine bone marrow damage was also evaluated in vivo. Again, the combined fractions shown a significant increase donor chimerism in bone marrow at 6 month post-transplant with 5x's the level of engraftment compared to irradiation control. The 10k and 100-10k fractions showed intermediate healing. Exposure to vesicles in irradiated FDC-P1 cells downregulated phosphorylated H2AX, the DNA damage marker and decreased apoptosis marker, PARP cleavage part. We further investigated the effect of murine marrow MSC-derived vesicles on the gene expression in peripheral blood cells from radiation damaged mice. 28 genes were altered by irradiation, 22 showed partial or complete reversal of these alterations. The characterization of three different fractions of vesicles derived from murine bone marrow cells and human MSC were evaluated by miRNA realtime PCR array (total 750 miRNA). Several MSC-EV associated miRNA responsible for the reversal of radiation damage were evaluated. Our preliminary data shown that miR-221, miR654-3p and miR210-5p could partially reverse radiation damage in FDC-P1 cells There are different biological effects in different extracellular vesicles populations. For reversal of radiation toxicity the most effective vesicle population would include both smaller (exosomes) and larger vesicles (microvesicles). Acknowedgements: This work was support by the NIH grant 5UH2TR00080 Disclosures No relevant conflicts of interest to declare.

Background: Hematopoietic stem cells (HSCs) have tremendous self-renewal and differentiation capacity. The majority of murine hematopoietic stem cell studies have focused on rare purified populations of HSCs, conventionally described as negative for lineage-specific markers and positive for particular cell surface epitope profiles, including c-Kit, Sca-1, and CD150. However, our data indicate that such purifications lead to the loss of a significant population of actively cycling marrow cells with long-term multi-lineage stem cell potential. In the studies presented here, we tested the hypothesis that this discarded stem cell population lies, in part, within the lineage positive (Lin+) fraction of marrow. Methods: We flushed whole bone marrow (WBM) from B6.SJL mice and incubated it with allophycocyanin-tagged antibodies against erythroid (TER119), myeloid (CD11b, GR1), B-lymphoid (B220), or T-lymphoid (CD3, CD4, CD8) markers. Different doses of each specific Lin+ subset isolated by fluorescence activated cell sorting were competitively engrafted into lethally irradiated C57BL/6 host mice. At 1,3, and 6 months post-transplant, peripheral blood was analyzed for donor contribution to chimerism and lineage specificity. Results: Although typically considered to be without stem cell activity, we found that all Lin+ sub-fractions upon single sorting were able to contribute to marrow repopulation in competitive bone marrow transplants. For example, when lethally irradiated recipient mice received 3x105 C57BL/6J competitive whole bone marrow cells in combination with single-sorted GR1+ ± CD11b+ cells (2x106 cells/mouse), peripheral blood showed 15% donor chimerism at 6 months. Similarly, if single sorted CD3+ ±CD4+ ±CD8+ cells (70,000 cells/mouse), B220+ cells (1x106 cells/mouse), or Ter119+ cells (1x106 cells/mouse) were competitively engrafted with 3x105 C57BL/6 WBM cells, the donor Lin+ sub-fractions contributed to 2%, 15%, and 35% peripheral blood chimerism at 6 months post-transplant, respectively. This contribution was multi-lineage in all cases. When we performed double sorting of the Lin+ subsets, there was a dramatically reduced engraftment capacity between 1-6% donor chimerism for all subgroups. However, we do not think the loss of stem cell capacity with double sorting seen in these studies is due merely to the loss of classical hematopoietic stem cells (Lineage-/stem cell marker+). In our earlier studies, we showed that the total Lin+ population contains long-term multi-lineage engraftment capacity due almost entirely to actively cycling cells. Therefore, if the engraftment capacity within the single sorted Lin+ sub-fractions was due solely to the presence of classical HSCs lost with double sorting, the engraftment capacity found within the Lin+ compartment should be due only to quiescent cells in keeping with the cell cycle status of engrafting highly purified stem cells. Conclusions: Based on these data, we predict that a cycling population of stem cells exists within this single sorted, Lin+ enriched fraction discarded with conventional HSC purification. Future studies are ongoing to further characterize the subsets of Lin+ cells that both remain Lin+ and are found to be Lin- upon double sorting. We will analyze these populations for engraftment capacity, concomitant stem cell marker expression and cell cycle status, in order to fully characterize the total stem cell potential within whole bone marrow that is not included in the purified HSC populations. Disclosures No relevant conflicts of interest to declare.

The NIH Extracellular RNA Communication Program’s initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.Keywords: extracellular vesicles; cell fate change; functional effects; renal; pulmonary heart disease; cancer(Published: 28 August 2015)Citation: Journal of Extracellular Vesicles 2015, 4: 27575 - http://dx.doi.org/10.3402/jev.v4.27575This paper is part of the Special Issue: Extracellular RNA Communication Consortium. More papers from this issue can be found at http://www.journalofextracellularvesicles.net

Objective Previous studies have demonstrated the production of various types of lung cells from marrow cells under diverse experimental conditions. Our aim was to identify some of the variables that influence conversion in the lung. Methods In separate experiments, mice received various doses of total-body irradiation followed by transplantation with whole bone marrow or various subpopulations of marrow cells (Lin −/+ , c-kit −/+ , Sca-1 −/+ ) from GFP + (C57BL/6-TgN[ACTbEGFP]1Osb) mice. Some were given intramuscular cardiotoxin and/or mobilized with granulocyte colony-stimulating factor (G-CSF). Results The production of pulmonary epithelial cells from engrafted bone marrow was established utilizing green fluorescent protein (GFP) antibody labeling to rule out autofluorescence and deconvolution microscopy to establish the colocaliztion of GFP and cytokeratin and the absence of CD45 in lung samples after transplantation. More donor-derived lung cells (GFP + /CD45 − ) were seen with increasing doses of radiation (5.43% of all lung cells, 1200 cGy). In the 900-cGy group, 61.43% of GFP + /CD45 − cells were also cytokeratin + . Mobilization further increased GFP + /CD45 − cells to 7.88% in radiation-injured mice. Up to 1.67% of lung cells were GFP + /CD45 − in radiation-injured mice transplanted with Lin − , c-kit + , or Sca-1 + marrow cells. Lin + , c-kit − , and Sca-1 − subpopulations did not significantly engraft the lung. Conclusions We have established that marrow cells are capable of producing pulmonary epithelial cells and identified radiation dose and G-CSF mobilization as variables influencing the production of lung cells from marrow cells. Furthermore, the putative lung cell–producing marrow cell has the phenotype of a hematopoietic stem cell.

The capacity of adult bone marrow cells to convert to cells of other tissues, referred to by many as stem cell plasticity, was the focus of the meeting in Providence entitled "Challenges in the Era of Stem Cell Plasticity". The meeting provided a showcase for the many impressive positive results on tissue restoration including the capacity of purified marrow stem cells to restore heart, skin, and liver function in impaired mice or humans. This area of research has become a center of controversy, although it is not clear why. Calls for clonality, robustness, and function have been shown to be erroneous or premature. A call for clonality (which has been shown nicely in one study) is meaningless on a predefined stem cell population which is intrinsically heterogeneous, as they all are. Robustness means nothing; it all depends on the details of the situation. Function on an organ level is, of course, the goal of many investigators and should not be raised as a limiting consideration. Lastly, fusion has been highlighted as undermining studies with adult stem cells. It, of course, does not. Fusion is simply a means to a final goal, which occurs in certain settings of marrow conversions (transdifferentiation) and not in others. We hypothesize that the conversion phenomena may, in fact, be due to one or several marrow stem cells with broad differentiation potential which can be expressed when the cell is placed in an environment with the appropriate inductive signals. Furthermore, initial events may be relatively rare and significant conversion numbers may be obtained with massive or ongoing selection. Fusion appears in an initial mechanism in some cases and not in others. Overall, the therapeutic potential of adult marrow stem cells is very intriguing, and successful use therapeutically will probably depend on definition of the most appropriate transplant model and tissue injury.

Introduction: Extracellular vesicles (EVs) are membrane-bound, subcellular fragments that contain DNA, RNA, protein and lipids, and play an important role in intercellular communication. They have been implicated in myriad physiologic and pathologic processes within the hematopoietic system including thrombosis and leukemia, and as such, have tremendous therapeutic potential if manipulated appropriately. In order to fully harness their clinical potential, it will be important to more fully characterize the factors that modulate their biogenesis and function. Numerous factors are thought to influence EVs and their ability to communicate with target marrow cells, but little is known about how circadian oscillations alter EV function. Using a well-established model of lung-derived EV modulation of marrow phenotype, we report here our studies exploring the effects of circadian rhythm on the ability of lung-derived EVs to alter the transcriptome of bone marrow target cells. Methods: C57BL/6 mice were housed on a 12-hour light/dark schedule. For the first set of experiments, lungs, blood and bone marrow were harvested (6-16 mice/time) at three different circadian time-points (8,12 and 24 hours after light onset (HALOs). EVs were isolated from each tissue at each time-point and quantified by nanoparticle tracking analysis. Second, to determine if circadian rhythm influences the ability of lung-derived EVs to modulate the transcriptome of target whole bone marrow cells (WBM), lungs were harvested (3 mice/time-point x 2 experiments) at 4,12,16, and 24 hours after light onset and co-cultured, across a cell-impermeable membrane, with murine WBM. The WBM was harvested after 24hrs in co-culture and analyzed for the induction of pulmonary specific mRNA expression by RT-PCR. Finally, to determine if altering the circadian timepoint of the target whole bone marrow influenced the ability of EVs to modulate marrow phenotype, lungs harvested from mice sacrificed at HALO 2 were cultured for 48hrs. EVs were isolated from the lung-conditioned media by ultracentrifugation. WBM was then harvested at different circadian time-points (HALO 4, 8, 12, 16, 20, and 24; 3 mice/time-point x 2 experiments), cultured with or without the lung-derived EVs for 24hrs, and then analyzed for pulmonary-specific mRNAs by RT-PCR. Results: There were changes in EV quantity related to circadian timepoint. There were significantly increased numbers of EVs generated from lungs harvested at HALO 24 and HALO 8 when compared to HALO 16. Similarly, marrow harvested at HALO 8 had significantly increased number of EVs as compared to WBM harvested at HALO 16 and 24. In addition to circadian-related differences in EV quantity, there were clear oscillations in the expression patterns of pulmonary specific mRNAs when both the circadian timepoint of the lung donor and the circadian timepoint of the recipient marrow cells were altered. If lung was harvested at HALO 12 or HALO 16, the co-cultured WBM showed significantly increased expression of pulmonary-specific mRNAs when compared to WBM co-cultured with lung harvested at HALO 4 or HALO 24. Likewise, when WBM harvested at different circadian time-points was incubated with lung-derived EVs, there was significantly increased expression of surfactant A, surfactant B, and aquaporin 5 when WBM was harvested at HALO 16 when compared to HALO 12 or HALO 24. Conclusions: Based on these data, we conclude that circadian rhythm is likely an important modulator of EV-mediated intercellular communication with whole bone marrow cells and warrants more detailed study. Disclosures No relevant conflicts of interest to declare.

The study of highly purified hematopoietic stem cells (HSCs) has dominated the field of hematopoietic stem cell biology. It is widely believed that the true stem cell population lies within the Lineage negative (Lin-) population, further sub-fractionated using positive and negative selection for surface markers such as c-Kit, Sca-1, CD150, CD41, CD48, and CD34. It is research on these highly purified subsets of HSCs that forms the foundation for almost all our knowledge of HSCs, and has led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to near homogeneity. In contrast, we have shown that a large percentage of long-term multi-lineage marrow repopulating cells in whole bone marrow (WBM) are actively cycling, that these cycling stem cells are lost during conventional HSC isolation, and that they can be found, in part, within the discarded Lineage positive (Lin+) population. Here we present data further characterizing the stem cell potential in the Lin+ fraction. We incubated WBM from B6.SJL mice with fluorescently tagged antibodies directed against TER119, B220, or T-cell markers (CD3, CD4, CD8), isolated the distinct Lin+ subsets by FACS, and then competitively engrafted each Lin+ subset into lethally irradiated C57BL/6 host mice. Donor chimerism and lineage specificity of donor cells in peripheral blood were analyzed by flow cytometry at 3 months. Although classically considered devoid of stem cell activity, we found that, when competed against equal numbers of C57BL/6 WBM, the TER119+ and B220+ B6.SJL donor cells contributed to 33% and 13% of the peripheral blood chimerism, respectively. In both cases, the engraftment was multi-lineage. When 70,000 T cell marker+ donor cells were competed with 300,000 C57BL/6 WBM, the donor cells contributed up to 1.6% of the peripheral blood multi-lineage chimerism. Given the size of the Lin+ fraction in WBM, such chimerism indicates a significant stem cell potential within this typically discarded population. Further time-points, secondary transplants and limited dilution studies are in progress to further define the prevalence and potency of this stem cell population. We have been testing mechanisms governing the loss of this stem cell population during HSC purification. First, we have previously shown that bulk Lin+ engraftment potential is due to cycling stem cells. We hypothesize that fluctuations in surface epitope expression with cell cycle transit render this population difficult to isolate with antibody-mediated strategies that rely on stable epitope expression. To begin testing this, we tracked the fluctuation of stem cell markers on Lin- cells in vitro. We isolated Lin- cells that were also negative for the stem cell markers c-Kit and Sca-1, placed them in liquid culture and, 18 hours later, re-assessed for stem cell marker expression by flow cytometry. We found that, although initially stem cell marker negative, up to 6%, 14%, and 2% of the Lin-/stem cell marker negative cells became positive for c-Kit alone, Sca-1 alone, or both c-Kit and Sca-1 expression, respectively. We are currently testing this population for a correlation between gain of c-Kit- and Sca-1 expression and stem cell function. Second, it is possible that there is a distinct subset of HSCs that are positive for both Lin+ markers and stem cell markers with stable stem cell capacity and that these distinct stem cells are thrown out in the process of lineage depletion. To begin testing this hypothesis, we have simultaneously stained WBM with antibodies directed against the Lin+ markers and conventional stem cell markers. Our preliminary data indicate that each Lin+ fraction tested to date has a subpopulation that is also positive for c-Kit and Sca-1. For example, 21% of CD3+ cells, 6.2% of CD4+ cells, 2.26% of CD8+ cells, 0.5% of B220+, and 0.45% of TER119+ cells express both c-Kit and Sca-1. We suspect these two populations have distinct functional phenotypes and experiments characterizing the molecular phenotype and engraftment capacity of these subpopulations are ongoing. In sum, our data indicate that stem cell purification skews isolation towards a small population of quiescent stem cells, underrepresenting a potentially large pool of actively cycling HSCs that are found within the Lin+ fraction. These data underscore the need to re-evaluate the total hematopoietic stem cell potential in marrow on a population level. Disclosures No relevant conflicts of interest to declare.

Adult marrow hematopoiesis has been felt to represent a classical hierarchical system with a primitive stem cell progressively differentiating into hematopoietic cells. Colony-forming unit spleen was the initial stem cell and there followed definition of a wide variety of progenitors with different lineage potentials. Purification studies indicated that the critical stem cells were lineage negative and Sca-1+c-kit+CD150+.1 Our studies over the past decade indicate that the phenotype of the stem cell changes reversibly with cell cycle transit. Characteristics showing such changes include long-term multilineage engraftment into irradiated mice, differentiation, homing to marrow, expression of adherence proteins and global gene expression, progenitor phenotype and uptake of extracellular vesicles. It was demonstrated that most stem cells are discarded during the separation. The final purified stem cell is in fact dormant, but the vast majority of stem cells that are discarded (over 90%) are in active cell cycle.2 In these studies it was shown that cells in S/G2/M accounted for over 50% of long-term engraftment of whole unseparated marrow and that the classical purified lineage-negative Sca-1-c-kit+CD150+ stem cell rapidly progressed through the cell cycle so that after 48 h exposure to in vivo bromodeoxyuridine up to 80% of the ‘dormant' cells incorporated bromodeoxyuridine. These data indicated that most mature marrow stem cells were discarded with the purification and that these cells were virtually all proliferating. Studies on the different fractions of a purification indicated that the vast majority of adult marrow stem cells were present in the lineage-positive and lineage-negative populations and that these cells were cycling. Thus, the true marrow stem cells rapidly transited the cell cycle and were continually changing phenotype; they could not be ‘purified' by assessing a set number of epitopic markers. The bulk of studies on murine marrow stem cells has been carried out on ‘purified stem cells' and thus have been addressing an irrelevant population of cells. These studies need to be readdressed. Further heterogeneity of adult marrow stem cells is introduced by considerations of interactions of these cells with extracellular vesicles.3 Studies on lineage-negative Sca-1+ progenitor/stem cells traversing the cell cycle under cytokine stimulation in liquid culture indicated that vesicle uptake varied with both the cell cycle state of the target cell and the nature of treatment of the originator lung cells (irradiation or no irradiation). Thus the adult marrow stem cell continually alters phenotype with cell cycle transit and this is further impacted upon by interactions with vesicles from a variety of cell types. The cell phenotype induced by vesicle exposure appears to be a stable epigenetic change induced by an RNase-sensitive agent that is not messenger RNA, presumably a non-coding RNA species. These observations suggest unique strategies for determination of cell fate and restoration of damaged cells. These data also suggest that while cell populations may be stable at the tissue or population level they are not stable at the individual cell level.

Abstract 4480 Objective Pulmonary complications are common in bone marrow transplant (BMT) recipients and often of an infectious etiology or attributed to intensive conditioning regimens. Cases of pulmonary arterial hypertension have been described in the setting of autologous BMT but are believed to be the result of acquired infections or previously-administered chemotherapy. Whether transplanted marrow cells themselves are toxic to the recipient's lungs is unknown. Methods To address this, non-irradiated female BALB/C mice were transplanted with 5-6×107 whole bone marrow (WBM) cells from male BALB/C mice daily for four days on week zero, then again on week eight (4.5×108 cells total/mouse), or an equal volume of diluent (control). On week 24, transplanted mice received 1000 cGy of chest-only radiation or no radiation. On week 32, histochemical and immunohistochemical analyses were performed on recipient's lungs. Results Bone marrow chimerism was not significantly different in the irradiated and non-irradiated cohorts at the time of sacrifice (average 45.11+6.25% Y chromosome+, all mice). Recipient lungs contained few non-hematopoietic donor marrow-derived cells. These cells were exclusively epithelial (Y+/cytokeratin+), primarily type II pneumocytes (Y+/prosurfactant C+), while no endothelial (Y+/von Willebrand+) or vascular smooth muscle (Y+/alpha-actin+) cells were identified. Irradiated and non-irradiated cohorts had similar quantities of these cells (0.80+0.22% vs. 0.51+0.08% Y+/cytokeratin+; 0.37+0.08% vs. 0.32+0.12% Y+/prosurfactant C+, p>0.2). Pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were significantly increased in the non-irradiated cohort compared with controls and these ratios were further increased in the irradiated cohort (141+5.75%, 161+5.34% of control, p Conclusions These data indicated that marrow cell infusion alone results in pulmonary vascular remodeling and this effect is augmented by radiation injury. These changes are independent of transplanted marrow cell conversion to pulmonary vascular endothelial, smooth muscle cells. These studies suggest that transplanted cells may be lung toxic entities in the setting clinical BMT. Disclosures: No relevant conflicts of interest to declare.

Abstract 5190 Rationale: Circulating endothelial cell-derived microparticles (MP) are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. We have shown that lung-derived MP induce marrow cells to express lung-specific mRNA, protein. Objectives: Determine if lung, plasma-derived MP (LMP, PMP) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodeling in PAH. Methods and Main Results: LMP, PMP isolated from monocrotaline (MCT)- or vehicle-treated mice were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodeling were assessed by RV-to-body weight (RV/BW), blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT vs. vehicle-injected mice (1. 99+0. 09vs. 1. 04+0. 09mg/g; 0. 159+0. 002vs. 0. 062+0. 009%). MP from MCT and vehicle-treated mice were quantitatively similar; however, MCT-LMP had higher endothelial cell mRNA expression and higher expression of mRNAs of proteins known to be abnormally expressed in PAH vs. vehicle-LMP. RV/BW, WT/D ratios were higher in mice injected with MCT-MP vs. mice injected with vehicle-MP (1. 63+0. 09vs. 1. 08+0. 09mg/g; 0. 113+0. 02vs. 0. 056+0. 01%). Lineage-depleted bone marrow cells co-cultured with MCT-MP and bone marrow cells isolated from MCT-MP infused mice had higher endothelial progenitor cell gene expression vs. cells co-cultured with vehicle-MP or cells isolated from vehicle MP infused mice. Conclusions: MP from MCT-injured mice have increased expression of growth factors implicated in PAH pathogenesis and induce RV hypertrophy, pulmonary vascular remodeling in healthy mice. Circulating MP may contribute to the development of MCT-induced pulmonary hypertension by inducing a pathologic phenotype in pulmonary vascular endothelial and endothelial progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 2308 Hematopoietic stem cell biologists have amassed a tremendous depth of knowledge about the biology of the marrow stem cell over the past few decades, facilitating invaluable basic scientific and translational advances in the field. Most of the studies to date have focused on highly purified populations of marrow cells, with emphasis placed on the need to isolate increasingly restricted subsets of marrow cells within the larger population of resident bone marrow cells in order to get an accurate picture of the true stem cell phenotype. Such studies have led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to homogeneity. However, work from our laboratory, focusing on the stem cell potential in un-separated whole bone marrow (WBM), supports an alternate view of marrow stem cell biology in which a large population of marrow stem cells are actively cycling, continually changing phenotype with cell cycle transit, and therefore, cannot be purified to homogeneity. Our studies separating WBM into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposing WBM to tritiated thymidine suicide followed by competitive engraftment into lethally irradiated mice revealed that over 50% of the long-term multi-lineage engraftment potential in un-separated marrow was due to cells in S/G2/M. This is in stark contrast to studies showing that highly purified stem cell populations such as LT-HSC (Lineage–c-kit+sca-1+flk2−) engraft predominantly when in G0. Additionally, by performing standard isolation of a highly purified population of stem cells, SLAM cells (Lineage–c-kit+sca-1+flk2−CD150+CD41−CD48−), and testing the engraftment potential of different cellular fractions created and routinely discarded during this purification process, we found that 90% of the potential engraftment capacity in WBM was lost during conventional SLAM cell purification. Incubation of the Lineage-positive and Lineage-negative fractions with tritiated thymidine, a DNA analogue which selectively kills cells traversing S-phase, led to dramatic reductions in long-term multi-lineage engraftment potential found within both cellular fractions (over 95% and 85% reduction, respectively). This indicates that the discarded population of stem cells during antibody-based stem cell purification is composed largely of cycling cells. In sum, these data strongly support that 1) whole bone marrow contains actively cycling stem cells capable of long-term multi-lineage engraftment, 2) these actively cycling marrow stem cells are lost during the standard stem cell purification strategies, and 3) the protean phenotype of actively cycling cells as they transit through cell cycle will render cycling marrow stem cells difficult to purify to homogeneity. Given the loss of a large pool of actively cycling HSC during standard stem cell isolation techniques, these data underscore the need to re-evaluate the total hematopoietic stem cell pool on a population level in addition to a clonal level in order to provide a more comprehensive study of HSC biology. Disclosures: No relevant conflicts of interest to declare.

e21086 Background: Microvesicles are subcellular entities that are derived from a variety of cells and contain DNA, RNA and protein. We have previously shown that microvesicles isolated from murine...

e21092 Background: We have previously shown that lung-derived microvesicles (MVs) can enter target marrow cells, resulting in increased levels of lung-specific mRNAs. Marrow which has been exposed ...

Abstract 4803 We have previously shown that murine bone marrow cells co-cultured opposite murine lung cells, but separated from them by a cell-impermeable membrane, express pulmonary epithelial cell-specific mRNAs, including Surfactants A-D, Aquaporin-5 and Clara Cell Specific Protein. This effect appears to be enhanced when the lungs used in co-culture are harvested from previously-irradiated mice. We have also shown that lung cells release membrane-bound particles called microvesicles which contain lung cell-specific proteins and mRNA. Microvesicles are capable of entering marrow cells in culture and inducing lung-specific mRNA and protein expression in marrow cells that have internalized them (Aliotta JM, et al, Stem Cells, 2007). Currently, it is not known how microvesicles gain entry into marrow cells and if this process involves the binding of specific adhesion proteins present on the marrow cells and/or the surface of microvesicles. Microvesicles derived from sources other than the lung have been described as expressing various adhesion proteins which facilitate their entry into target cells. In order to determine the presence of adhesion proteins on lung-derived microvesicles, we ultracentrifuged (100,000 g) cell-free conditioned media made from the lungs of C57BL/6 mice. Pelleted material was labeled with one of several directly-conjugated antibodies to an adhesion protein. Microvesicles which were positive for the adhesion protein of interest were identified by flow cytometry and quantified relative to its isotype control. Adhesion proteins that were found to be present on lung-derived microvesicles included CD29 (β1 integrin), CD41 (α2b integrin), CD49e (α5 integrin), CD49f (α6 integrin), CD51 (αv integrin), CD54 (ICAM-1), CD61 (β3 integrin), CD62L (L-selectin), CD81 (TAPA-1), CD107a (LAMP-1), CD154 (CD40 ligand), CD184 (CXCR4) and CD309 (VEGFR2). Those not present included CD9 (p24), CD11b (Mac-1), CD18 (β2 integrin), CD31 (PECAM-1), CD49d (α4 integrin), CD62P (P-selectin) and CD162 (PSGL-1). Lung-derived microvesicles isolated from mice that had been exposed to 500 centigrey total body irradiation five days prior to sacrifice were enriched with certain populations of adhesion protein positive microvesicles compared to those isolated from non-irradiated mice. These populations included ICAM-1 (78% increase), TAPA-1 (45% increase), CD40 ligand (108% increase) and VEGFR2 (136% increase) positive microvesicles. We are attempting to determine if lung-derived microvesicle entry into marrow cells is mediated by interactions with these adhesion proteins. These data indicate that adhesion proteins are present on lung-derived microvesicles. It is possible that differences in mRNA expression seen in marrow cells exposed to microvesicles derived from irradiated and non-irradiated lung in be related to qualitative and quantitative differences in those microvesicles. Disclosures: No relevant conflicts of interest to declare.

Abstract 4799 We have previously shown that microvesicles derived from irradiated murine lung cells enter murine bone marrow cells and induce expression of the pulmonary epithelial cell-specific mRNAs Surfactants A-D (Sp-A-D), Aquaporin-5 (Aq-5) and Clara Cell Specific Protein (CCSP). The early mRNA increases are due to direct delivery of mRNA and due to the transfer of a transcription factor to marrow cells (Aliotta JM, et al, Exp Hematol, 2010). The modulated marrow cells produce pulmonary epithelial cell-specific protein and have an enhanced ability to convert into pulmonary epithelial cells after transplantation into lethally-irradiated mice. In the present studies, we evaluated the stability of pulmonary epithelial cell-specific mRNA expression in cytokine-stimulated liquid culture and after in vivo transplantation into lethally-irradiated mice. Whole bone marrow cells isolated from C57BL/6 mice were cultured for seven days opposite C57BL/6 lung fragments, but separated from them by a 0.4 micron cell-impermeable membrane. Marrow cells were then either infused into lethally-irradiated C57BL/6 mice or established in culture with DMEM supplemented with IL-3, IL-6, IL-11 and steel factor. Engrafted mice were sacrificed six weeks after transplantation and real-time RT-PCR was carried out on marrow, liver, spleen and thymus tissue. Expression of Sp-A, B and D, CCSP and Aq-5 were variably seen in all tissues and increases over control ranged from 12-fold to 58-fold. Liquid culture was analyzed every two weeks out to 12 weeks and high levels of expression of different pulmonary epithelial cell-specific mRNAs and proteins (by western blot and Immunohistochemical staining) were observed. In order to determine the mechanism for the increased expression in culture, we established co-cultures of Fischer-344 rat lung opposite C57BL/6 mouse bone marrow cells and, using species-specific primers, determined whether the increased pulmonary epithelial cell-specific mRNA expression at each time point of secondary cytokine-supported culture was rat or mouse-specific. In every instance, the pulmonary epithelial cell-specific mRNA which was detected was mouse-specific, not rat-specific, and thus the long-term expression of lung-specific mRNA in mouse marrow was due to induced transcription by the mouse marrow cells. These data indicate that lung-derived microvesicles induce long-term stable epigenetic changes in marrow cells and suggests that the genetic changes induced by tissue microvesicles in neighboring cells may be biologically important. Disclosures: No relevant conflicts of interest to declare.

Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. Standard hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum and believe that sensitivity to cytokines change as a stem/progenitor cells goes through cell cycle transit. We previously have shown that stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegativerhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. We have demonstrated differentiation hotspots on a cell cycle continuum (Exp Heme35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine LRH marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around giving an inductive signal after 32-hours in primary culture; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over half hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. Stem cell differentiation hotspots may eventually be utilized to alter repopulation kinetics after bone marrow transplantation improving recovery time of platelets and neutrophils, translating into improved outcomes.

We have previously demonstrated that murine bone marrow cells co-cultured with lung opposite a cell-impermeable membrane express high levels of lung cell-specific genes (Aliotta et al, Stem Cells, 2007;25(9):2245–56). Greater changes in gene expression were noted in marrow cells co-cultured with radiation-injured lung. A factor smaller than the pore size of the cell-impermeable membrane (0.4μm) is responsible for these changes as cell-free conditioned media (CM) had a similar effect on co-cultured marrow cells. Lung-derived microvesicles were found to enter marrow cells in culture and may be among the factors responsible for these phenotypic changes. We wished to determine if these observations could be generalized to co-cultures using other murine organs and whether these changes in gene expression are tissue-specific. Whole bone marrow (WBM) cells were isolated from C57BL/6 mice and co-cultured with lung, liver, heart and WBM cells from C57BL/6 mice exposed to 1200 centigrey (cGy) of total body irradiation (TBI) or no radiation. Control WBM cells were co-cultured with no tissue (control). Co-cultured WBM cells were analyzed 7 or 14 days later by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). In addition, cell-free CM made from the same organs was co-cultured for 7 or 14 days with WBM cells which were then analyzed by RT-PCR. Alternatively, CM was analyzed for the presence of microvesicles by electron microscopy (EM) of ultracentrifuged (UCF) pelleted material. WBM co-cultured only with lung had increased gene expression of surfactant proteins A (Sp-A) and C (Sp-C, 89 and 334-fold increase vs. control, respectively) whereas WBM co-cultured only with brain had increased gene expression of Glial Fibrillary Acidic Protein (GFAP, 4.6-fold increase vs. control). Slight increases in Albumin expression were seen in all co-culture groups but expression was markedly elevated in WBM co-cultured with liver (162,657-fold increase vs. control). Expression of heart-specific markers, including Troponin I and T2, was seen in WBM co-cultured with heart but these levels were not significantly different from those of other co-culture groups. Radiation injury augmented expression of certain genes in co-cultured WBM, including Sp-A (1019 vs. 89-fold increase) in lung co-cultures and GFAP (24 vs. 4.6-fold increase) in brain co-cultures. WBM co-cultured with CM from all organs demonstrated similar changes in gene expression. In addition, pelleted material from UCF CM contained RNA that was specific to the tissue from which the CM was made. EM of UCF CM demonstrated numerous membranebound particles 50–200nm in size that were typical of microvesicles in appearance. These data suggest that changes seen in gene expression of co-cultured WBM are largely tissue-specific, depending on the tissue they are co-cultured with. Microvesicles released by various tissues in co-culture may be among the mediators responsible for the changes seen in WBM gene expression.

We have previously described that RNA-filled microvesicles are shed from lung cells and are capable of entering whole bone marrow (WBM) cells in culture (Aliotta et al, Stem Cells, 2007;25(9):2245–56). WBM cells cultured with these lung-derived microvesicles appear to express lung cell-specific genes after 7 days in culture. However, it is not known if the lung cell-specific mRNA detected in these cells represents de novo transcription or previously transcribed mRNA carried into the cell via microvesicles. To address this issue, we co-cultured WBM cells extracted from C57BL/6 mice and co-cultured them opposite lung from C57BL/6 mice exposed to 500 centigrey (cGy) of total body irradiation (TBI) or no radiation. The two cell types were separated by a cell-impermeable membrane (pore size, 0.4μm). After 7 days of co-culture, WBM cells were removed, extensively washed and cultured alone for an additional 36 hours, with or without the transcriptional blocking agent Actinomycin-D (Act-D, 10 ug/ml). Control WBM cells were co-cultured without lung and not exposed to Act-D (control). WBM cells were then analyzed for the presence of a variety of hematopoietic and lung-specific markers by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). WBM cells co-cultured with or without lung expressed high levels of hematopoietic markers including Sca-1, c-kit and CD45. This expression was nearly or completely eliminated after exposure to Act-D in culture. Expression of the surfactant protein B (Sp-B) gene was also significantly decreased in WBM co-cultured with lung (irradiated or non-irradiated) after exposure to Act-D (mean ± SE, from 16±4.9 to 1.1±0.4-fold increase vs. control, p=0.012). Interestingly, exposure to Act-D resulted in an increase in the expression of other lung-specific genes, including surfactant protein C (Sp-C) and clara cell specific protein (CCSP), in WBM co-cultured with lung (from 165±103 to 3,700±1,625 and 2±0.9 to 93±38-fold increases vs. control, p

A body of work in the literature has shown that after marrow transplantation into irradiated mice, there appear cells in the lung with an epithelial lung cell phenotype, but with markers of the donor marrow cells. After transplantation of green-fluorescent protein positive marrow cells into lethally irradiated mice, we have previously shown that there are from 3–5% non-hematopoietic cells bearing the donor marrow markers in the lung, most of these being cytokeratin positive (Aliotta et al., Exp Hematol., 34(2):230–41, 2006). We have utilized a cell impermeable double culture chamber to co-culture irradiated and non-irradiated lung across from marrow cells. With 2 or 7 days of co-culture, the marrow cells express the lung-specific mRNAs surfactant proteins B and C and Clara Cell specific protein at high levels. This phenomenon is most pronounced when the lung has been exposed to 500 cGy 5 days prior to initiating co-culture, although it is also seen with non-irradiated lung. Conditioned media from lung exerts the same effects and RNase treatment of the conditioned media markedly decreased this effect. Ultracentrifugation of the conditioned media pellets the converting activity, which appears to reside in microvesicles. These microvesicles were demonstrated to enter marrow stem cells and to enhance their capacity to convert to lung epithelial cells after transplantation. Most recently, we have also shown that the capacity to enter marrow cells varies both with the cell cycle status of lineage negative Sca-1+ murine marrow cells and with the source of the microvesicles, either from irradiated or normal lung. Altogether, these data indicate that microvesicle transfer from damaged tissue may be the basis of some forms of marrow plasticity, which have been previously reported.