Mesenchymal Stem Cell Extracellular Vesicles Reverse Sugen/Hypoxia Pulmonary Hypertension in Rats.

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 < 0.01), and increased numbers of peripheral blood vessels (11.8 ± 0.66 vs. 6.9 ± 0.57 vessels per field; P < 0.001). Mesenchymal stem cell extracellular vesicles are effective at preventing and reversing pulmonary hypertension in Sugen/hypoxia pulmonary hypertension and may offer a new approach for the treatment of pulmonary arterial hypertension.

Concise reviews: A stem cell apostasy: a tale of four H words.

The field of hematopoietic stem cell (HSC) biology has become increasingly dominated by the pursuit and study of highly purified populations of HSCs. Such HSCs are typically isolated based on their cell surface marker expression patterns and ultimately defined by their multipotency and capacity for self-generation. However, even with progressively more stringent stem cell separation techniques, the resultant HSC population remains heterogeneous with respect to both self-renewal and differentiation capacity. Critical studies on unseparated whole bone marrow have definitively shown that long-term engraftable HSCs are in active cell cycle and thus continually changing phenotype. Therefore, they cannot be purified by current approaches dependent on stable surface epitope expression because the surface markers are continually changing as well. These critical cycling cells are discarded with current stem cell purifications. Despite this, research defining such characteristics as self-renewal capacity, lineage-commitment, bone marrow niches, and proliferative state of HSCs continues to focus predominantly on this small subpopulation of purified marrow cells. This review discusses the research leading to the hierarchical model of hematopoiesis and questions the dogmas pertaining to HSC quiescence and purification.

A new stem cell biology: the continuum and microvesicles.

The hierarchical models of stem cell biology have been based on work first demonstrating pluripotental spleen-colony-forming units, then showing progenitors with many differentiation fates assayed in in vitro culture; there followed the definition and separation of "stem cells" using monoclonal antibodies to surface epitopes and fluorescent-activated cell characterization and sorting (FACS). These studies led to an elegant model of stem cell biology in which primitive dormant G0 stem cells with tremendous proliferative and differentiative potential gave rise to progressively more restricted and differentiated classes of stem/progenitor cells, and finally differentiated marrow hematopoietic cells. The holy grail of hematopoietic stem cell biology became the purification of the stem cell and the clonal definition of this cell. Most recently, the long-term repopulating hematopoietic stem cell (LT-HSC) has been believed to be a lineage negative sca-1+C-kit+ Flk3- and CD150+ cell. However, a series of studies over the past 10 years has indicated that murine marrow stem cells continuously change phenotype with cell cycle passage. We present here studies using tritiated thymidine suicide and pyronin-Hoechst FACS separations indicating that the murine hematopoietic stem cell is a cycling cell. This would indicate that the hematopoietic stem cell must be continuously changing in phenotype and, thus, could not be purified. The extant data indicate that murine marrow stem cells are continually transiting cell cycle and that the purification has discarded these cycling cells. Further in vivo BrdU studies indicate that the "quiescent" LT-HSC in G0 rapidly transits cycle. Further complexity of the marrow stem cell system is indicated by studies on cell-derived microvesicles showing that they enter marrow cells and transcriptionally alter their cell fate and phenotype. Thus, the stem cell model is a model of continuing changing potential tied to cell cycle and microvesicle exposure. The challenge of the future is to define the stem cell population, not purify the stem cell. We are at the beginning of elucidation of quantum stemomics.

The universal stem cell.

Current dogma is that there exists a hematopoietic pluripotent stem cell, resident in the marrow, which is quiescent, but with tremendous proliferative and differentiative potential. Furthermore, the hematopoietic system is essentially hierarchical with progressive differentiation from the pluripotent stem cells to different classes of hematopoietic cells. However, results summarized here indicate that the marrow pluripotent hematopoietic stem cell is actively cycling and thus continually changing phenotype. As it progresses through cell cycle differentiation potential changes as illustrated by sequential changes in surface expression of B220 and GR-1 epitopes. Further data indicated that the potential of purified hematopoietic stem cells extends to multiple other non-hematopoietic cells. It appears that marrow stem cells will give rise to epithelial pulmonary cells at certain points in cell cycle. Thus, it appears that the marrow "hematopoietic" stem cell is also a stem cell for other non-hematopoietic tissues. These observations give rise to the concept of a universal stem cell. The marrow stem cell is not limited to hematopoiesis and its differentiation potential continually changes as it transits cell cycle. Thus, there is a universal stem cell in the marrow which alters its differentiation potential as it progresses through cell cycle. This potential is expressed when it resides in tissues compatible with its differentiation potential, at a particular point in cell cycle transit, or when it interacts with vesicles from that tissue.

Differentiation Epitopes Define Hematopoietic Stem Cells and Change with Cell Cycle Passage.

Hematopoietic stem cells express differentiation markers B220 and Gr1 and are proliferative. We have shown that the expression of these entities changes with cell cycle passage. Overall, we conclude that primitive hematopoietic stem cells alter their differentiation potential with cell cycle progression. Murine derived long-term hematopoietic stem cells (LT-HSC) are cycling and thus always changing phenotype. Here we show that over one half of marrow LT-HSC are in the population expressing differentiation epitopes and that B220 and Gr-1 positive populations are replete with LT-HSC after a single FACS separation but if subjected to a second separation these cells no longer contain LT-HSC. However, with second separated cells there is a population appearing that is B220 negative and replete with cycling c-Kit, Sca-1 CD150 positive LT-HSC. There is a 3-4 h interval between the first and second B220 or GR-1 FACS separation during which the stem cells continue to cycle. Thus, the LT-HSC have lost B220 or GR-1 expression as the cells progress through cell cycle, although they have maintained the c-kit, Sca-1 and CD150 stem cells markers over this time interval. These data indicate that cycling stem cells express differentiation epitopes and alter their differentiation potential with cell cycle passage.

Targeting RUNX1 as a novel treatment modality for pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. Previously, we found that transcription factor RUNX1-dependent haematopoietic transformation of endothelial progenitor cells may contribute to the pathogenesis of PAH. However, the therapeutic potential of RUNX1 inhibition to reverse established PAH remains unknown. In the current study, we aimed to determine whether RUNX1 inhibition was sufficient to reverse Sugen/hypoxia (SuHx)-induced pulmonary hypertension (PH) in rats. We also aimed to demonstrate possible mechanisms involved. METHODS AND RESULTS: We administered a small molecule specific RUNX1 inhibitor Ro5-3335 before, during, and after the development of SuHx-PH in rats to investigate its therapeutic potential. We quantified lung macrophage recruitment and activation in vivo and in vitro in the presence or absence of the RUNX1 inhibitor. We generated conditional VE-cadherin-CreERT2; ZsGreen mice for labelling adult endothelium and lineage tracing in the SuHx-PH model. We also generated conditional Cdh5-CreERT2; Runx1(flox/flox) mice to delete Runx1 gene in adult endothelium and LysM-Cre; Runx1(flox/flox) mice to delete Runx1 gene in cells of myeloid lineage, and then subjected these mice to SuHx-PH induction. RUNX1 inhibition in vivo effectively prevented the development, blocked the progression, and reversed established SuHx-induced PH in rats. RUNX1 inhibition significantly dampened lung macrophage recruitment and activation. Furthermore, lineage tracing with the inducible VE-cadherin-CreERT2; ZsGreen mice demonstrated that a RUNX1-dependent endothelial to haematopoietic transformation occurred during the development of SuHx-PH. Finally, tissue-specific deletion of Runx1 gene either in adult endothelium or in cells of myeloid lineage prevented the mice from developing SuHx-PH, suggesting that RUNX1 is required for the development of PH. CONCLUSION: By blocking RUNX1-dependent endothelial to haematopoietic transformation and pulmonary macrophage recruitment and activation, targeting RUNX1 may be as a novel treatment modality for pulmonary arterial hypertension.

Intercellular transfer of proteins as identified by stable isotope labeling of amino acids in cell culture.

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.

Effect of dose, dosing intervals, and hypoxic stress on the reversal of pulmonary hypertension by mesenchymal stem cell extracellular vesicles.

RATIONALE: Mesenchymal stem cell extracellular vesicles (MSC EVs) reverse pulmonary hypertension, but little information is available regarding what dose is effective and how often it needs to be given. This study examined the effects of dose reduction and use of longer dosing intervals and the effect of hypoxic stress of MSC prior to EV collection. METHODS: Adult male rats with pulmonary hypertension induced by Sugen 5416 and three weeks of hypoxia (SuHx-pulmonary hypertension) were injected with MSC EV or phosphate buffered saline the day of removal from hypoxia using one of the following protocols: (1) Once daily for three days at doses of 0.2, 1, 5, 20, and 100 µg/kg, (2) Once weekly (100 µg/kg) for five weeks, (3) Once every other week (100 µg/kg) for 10 weeks, (4) Once daily (20 µg/kg) for three days using EV obtained from MSC exposed to 48 h of hypoxia (HxEV) or MSC kept in normoxic conditions (NxEV). MAIN RESULTS: MSC EV reversed increases in right ventricular systolic pressure (RVSP), right ventricular to left ventricle + septum weight (RV/LV+S), and muscularization index of pulmonary vessels ≤50 µm when given at doses of 20 or 100 µg/kg. RVSP, RV/LV+S, and muscularization index were significantly higher in SuHx-pulmonary hypertension rats treated once weekly with phosphate buffered saline for five weeks or every other week for 10 weeks than in normoxic controls, but not significantly increased in SuHx-pulmonary hypertension rats given MSC EV. Both NxEV and HxEV significantly reduced RVSP, RV/LV+S, and muscularization index, but no differences were seen between treatment groups. CONCLUSIONS: MSC EV are effective at reversing SuHx-pulmonary hypertension when given at lower doses and longer dosing intervals than previously reported. Hypoxic stress does not enhance the efficacy of MSC EV at reversing pulmonary hypertension. These findings support the feasibility of MSC EV as a long-term treatment for pulmonary hypertension.

Heterogeneity of non-cycling and cycling synchronized murine hematopoietic stem/progenitor cells.

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

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

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

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

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

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

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

Stem cell continuum: directed differentiation hotspots.

OBJECTIVE: The purpose of this study was to evaluate the technique of stem cell-directed differentiation in the context of cell-cycle position. The hypothesis was that stem cells would have different sensitivities to an identical inductive signal through cell-cycle transit and that this would affect the outcome of its progeny. MATERIALS AND METHODS: Differentiation of murine marrow lineage(negative)rhodamine-123(low-)Hoechst-33342(low) (LRH) stem cells was determined at different points in cell cycle under stimulation by thrombopoietin, flt3 ligand, and steel factor. LRH stem cells were subcultured in granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and steel factor at different points in cell cycle and differentiation determined 14 days later. RESULTS: There was a significant, reproducible, and pronounced reversible increase in differentiation to megakaryocytes in early S-phase and to nonproliferative granulocytes in mid S-phase. Megakaryocyte hotspots also were seen on a clonal basis. Elevations of the transcription factor FOG-1 were seen at the hotspot along with increases in Nfe2 and Fli1. CONCLUSIONS: We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell-cycle transit, suggesting that stem cell regulation is not based on the classic hierarchical model, but instead on a functional continuum. We propose that there is a tight linkage of commitment to a lineage and a particular phase of cell cycle. Thus, windows of vulnerability for commitment can open and close depending on the phase of cell cycle. These data indicate that stem cell differentiation occurs on a cell-cycle-related continuum with fluctuating windows of transcriptional opportunity.

Daily rhythms influence the ability of lung-derived extracellular vesicles to modulate bone marrow cell phenotype.

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.

SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity.

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using "Cre-loxP"-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.

Bone marrow production of lung cells: the impact of G-CSF, cardiotoxin, graded doses of irradiation, and subpopulation phenotype.

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.

Bone Marrow Endothelial Progenitor Cells Are the Cellular Mediators of Pulmonary Hypertension in the Murine Monocrotaline Injury Model.

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. Stem Cells Translational Medicine 2017;6:1595-1606.

Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication.

A rare subset of HIV-1-infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1-infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1-infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection.

Exosomes induce and reverse monocrotaline-induced pulmonary hypertension in mice.

AIMS: Extracellular vesicles (EVs) from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) induce PH in healthy mice, and the exosomes (EXO) fraction of EVs from mesenchymal stem cells (MSCs) can blunt the development of hypoxic PH. We sought to determine whether the EXO fraction of EVs is responsible for modulating pulmonary vascular responses and whether differences in EXO-miR content explains the differential effects of EXOs from MSCs and mice with MCT-PH. METHODS AND RESULTS: Plasma, lung EVs from MCT-PH, and control mice were divided into EXO (exosome), microvesicle (MV) fractions and injected into healthy mice. EVs from MSCs were divided into EXO, MV fractions and injected into MCT-treated mice. PH was assessed by right ventricle-to-left ventricle + septum (RV/LV + S) ratio and pulmonary arterial wall thickness-to-diameter (WT/D) ratio. miR microarray analyses were also performed on all EXO populations. EXOs but not MVs from MCT-injured mice increased RV/LV + S, WT/D ratios in healthy mice. MSC-EXOs prevented any increase in RV/LV + S, WT/D ratios when given at the time of MCT injection and reversed the increase in these ratios when given after MCT administration. EXOs from MCT-injured mice and patients with idiopathic pulmonary arterial hypertension (IPAH) contained increased levels of miRs-19b,-20a,-20b, and -145, whereas miRs isolated from MSC-EXOs had increased levels of anti-inflammatory, anti-proliferative miRs including miRs-34a,-122,-124, and -127. CONCLUSION: These findings suggest that circulating or MSC-EXOs may modulate pulmonary hypertensive effects based on their miR cargo. The ability of MSC-EXOs to reverse MCT-PH offers a promising potential target for new PAH therapies.

Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: a multifactorial process.

OBJECTIVE: Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the "robustness" of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow-to-muscle conversion. METHODS: We transplanted green fluorescence protein (GFP)-transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopoietic markers. RESULTS: Irradiation was essential to conversion, although whether by injury or induction of chimerism is not clear. Cardiotoxin- and, to a lesser extent, PBS-injected muscles showed significant number of GFP(+) muscle fibers, while uninjected muscles showed only rare GFP(+) cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent by G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57BL/6 and GFP to ROSA26 mice showed fusion of donor cells to recipient muscle. High numbers of donor-derived muscle colonies and up to 12% GFP(+) muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels that could be clinically significant in developing strategies for the treatment of muscular dystrophies. CONCLUSION: In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.