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.

Biodistribution of Mesenchymal Stem Cell-Derived Extracellular Vesicles in a Radiation Injury Bone Marrow Murine Model.

We have previously shown that injury induced by irradiation to murine marrow can be partially or completely reversed by exposure to human or murine mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). Investigation of the biodistribution of EVs in vivo is essential for understanding EV biology. In this study, we evaluated the DiD lipid dye labeled MSC-EV biodistribution in mice under different conditions, including different MSC-EV doses and injection schedules, time post MSC-EV injection, and doses of radiation. DiD-labeled MSC-EVs appeared highest in the liver and spleen; lower in bone marrow of the tibia, femur, and spine; and were undetectable in the heart, kidney and lung, while a predominant EV accumulation was detected in the lung of mice infused with human lung fibroblast cell derived EVs. There was significantly increased MSC-EV accumulation in the spleen and bone marrow (tibia and femur) post radiation appearing with an increase of MSC-EV uptake by CD11b+ and F4/80+ cells, but not by B220 cells, compared to those organs from non-irradiated mice. We further demonstrated that increasing levels of irradiation caused a selective increase in vesicle homing to marrow. This accumulation of MSC-EVs at the site of injured bone marrow could be detected as early as 1 h after MSC- EV injection and was not significantly different between 2 and 24 h post MSC-EV injection. Our study indicates that irradiation damage to hematopoietic tissue in the spleen and marrow targets MSC-EVs to these tissues.

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.

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.

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.

Homing and long-term engraftment of long- and short-term renewal hematopoietic stem cells.

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

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

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

Neutrophil depletion blocks early collagen degradation in repairing cholestatic rat livers.

Biliary obstruction results in a well-characterized cholestatic inflammatory and fibrogenic process; however, the mechanisms and potential for liver repair remain unclear. We previously demonstrated that Kupffer cell depletion reduces polymorphonuclear cell (neutrophil) (PMN) and matrix metalloproteinase (MMP)8 levels in repairing liver. We therefore hypothesized that PMN-dependent MMP activity is essential for successful repair. Male Sprague-Dawley rats received reversible biliary obstruction for 7 days, and the rat PMN-specific antibody RP3 was administered 2 days before biliary decompression (repair) and continued daily until necropsy, when liver underwent morphometric analysis, immunohistochemistry, quantitative RT-PCR, and in situ zymography. We found that RP3 treatment did not reduce Kupffer cell or monocyte number but significantly reduced PMN number at the time of decompression and 2 days after repair. RP3 treatment also blocked resorption of type I collagen. In addition, biliary obstruction resulted in increased expression of MMP3, MMP8, and tissue inhibitor of metalloproteinase 1. Two days after biliary decompression, both MMP3 and tissue inhibitor of metalloproteinase 1 expression declined toward sham levels, whereas MMP8 expression remained elevated and was identified in bile duct epithelial cells by immunohistochemistry. PMN depletion did not alter the hepatic expression of these genes. Conversely, collagen-based in situ zymography demonstrated markedly diminished collagenase activity following PMN depletion. We conclude that PMNs are essential for collagenase activity and collagen resorption during liver repair, and speculate that PMN-derived MMP8 or PMN-mediated activation of intrinsic hepatic MMPs are responsible for successful liver repair.

Dexamethasone alters the hepatic inflammatory cellular profile without changes in matrix degradation during liver repair following biliary decompression.

BACKGROUND: Biliary atresia is characterized by extrahepatic bile duct obliteration along with persistent intrahepatic portal inflammation. Steroids are standard in the treatment of cholangitis following the Kasai portoenterostomy, and were advocated for continued suppression of the ongoing immunologic attack against intrahepatic ducts. Recent reports, however, have failed to demonstrate an improved patient outcome or difference in the need for liver transplant in postoperative patients treated with a variety of steroid regimes compared with historic controls. In the wake of progressive liver disease despite biliary decompression, steroids are hypothesized to suppress inflammation and promote bile flow without any supporting data regarding their effect on the emerging cellular and molecular mechanisms of liver repair. We have previously shown in a reversible model of cholestatic injury that repair is mediated by macrophages, neutrophils, and specific matrix metalloproteinase activity (MMP8); we questioned whether steroids would alter these intrinsic mechanisms. METHODS: Rats underwent biliary ductal suspension for 7 d, followed by decompression. Rats were treated with IV dexamethasone or saline at the time of decompression. Liver tissue obtained at the time of decompression or after 2 d of repair was processed for morphometric analysis, immunohistochemistry, and quantitative RT-PCR. RESULTS: There was a dramatic effect of dexamethasone on the inflammatory component with the initiation of repair. Immunohistochemistry revealed a reduction of both ED1+ hepatic macrophages and ED2+Kupffer cells in repair compared with saline controls. Dexamethasone treatment also reduced infiltrating neutrophils by day 2. TNF-alpha expression, increased during injury in both saline and dexamethasone groups, was markedly reduced by dexamethasone during repair (day 2) whereas IL-6, IL-10, and CINC-1 remained unchanged compared with saline controls. Dexamethasone reduced both MMP8 and TIMP1 expression by day 2, whereas MMP9, 13, and 14 were unchanged compared with sham controls. Despite substantial cellular and molecular changes during repair, collagen resorption was the same in both groups CONCLUSION: Dexamethasone has clear effects on both the hepatic macrophage populations and infiltrating neutrophils following biliary decompression. Altered MMP and TIMP gene expression might suggest that steroids have the potential to modify matrix metabolism during repair. Nevertheless, successful resorption of collagen fibrosis proceeded presumably through other MMP activating mechanisms. We conclude that steroids do not impede the rapid intrinsic repair mechanisms of matrix degradation required for successful repair.

Hepatic macrophages promote the neutrophil-dependent resolution of fibrosis in repairing cholestatic rat livers.

BACKGROUND: Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. METHODS: Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl(3)) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. RESULTS: GdCl(3) treatment for either 24 or 48 hours before decompression reduced the numbers of ED2(+) Kupffer cells and ED1(+) inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl(3) pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl(3) treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. CONCLUSION: We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation.

Kupffer cells abrogate cholestatic liver injury in mice.

BACKGROUND & AIMS: Biliary obstruction and cholestasis can cause hepatocellular apoptosis and necrosis. Ligation of the common bile duct in mice provides an excellent model in which to study the underlying mechanisms. Kupffer cells play a key role in modulating the inflammatory response observed in most animal models of liver injury. This study was performed to determine the role of Kupffer cells in the injury attending cholestasis. METHODS: Mice were not treated or were rendered Kupffer cell-depleted by intravenous inoculation of multilamellar liposome-encapsulated dichloromethylene diphosphonate, the common bile duct was ligated and divided; sham-operated animals served as controls. Similarly, interleukin-6 (IL-6)-deficient and tumor necrosis factor-receptor-deficient mice underwent bile duct ligation (BDL) or sham operations. RESULTS: Serum alanine transaminase levels were increased in all BDL mice at 3 days after surgery, but were significantly higher in IL-6-deficient mice or mice rendered Kupffer cell-depleted before ligation. Histologic examination of BDL livers showed portal inflammation, neutrophil infiltration, bile duct proliferation, and hepatocellular necrosis. Photoimage analyses confirmed more necrosis in the livers of Kupffer cell-depleted and IL-6-deficient animals. Purified Kupffer cells derived from BDL animals produced more IL-6 in culture. Similarly, Kupffer cells obtained by laser capture microdissection from the livers of BDL mice expressed increased levels of IL-6 messenger RNA. Recombinant mouse IL-6 administered 1 hour before BDL completely reversed the increased liver damage assessed otherwise in Kupffer cell-depleted mice. CONCLUSIONS: These findings indicate that Kupffer cells abrogate cholestatic liver injury by cytokine-dependent mechanisms that include the production of IL-6.

Repair after cholestatic liver injury correlates with neutrophil infiltration and matrix metalloproteinase 8 activity.

BACKGROUND: Although timely surgical treatment of liver disease can interrupt inflammation and reduce fibrosis, the mechanisms of repair are unknown. We questioned whether these mechanisms of repair include changes in the inflammatory infiltrate and associated biological activity of matrix metalloproteinases (MMPs) 8 and 2. METHODS: Rats (n >or= 3) underwent biliary ductal suspension for 7 days followed by decompression. Livers were collected after 7 days of obstruction (d0) and after 2, 5, and 7 days of repair (d2, d5, d7, respectively), and assessed morphometrically for collagen, polymorphonuclear cells (PMNs), Kupffer cells (KCs), and inflammatory mononuclear phagocytes (MNPs). In situ zymography was performed by using fluorogenic substrates for MMP-8 and MMP-2 to spatially localize enzymatic activity. RESULTS: Cholestatic injury resulted in significantly elevated (P