Cyto B dependent and ouabain insensitive regulatory volume decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van't Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron x min(-1) x atm(-1). Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.     

Cyto B dependent and ouabain insensitive Regulatory Volume Decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco’s experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van’ t Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron · min⁻¹ · atm⁻¹. Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

Primary mesenchymal stem and progenitor cells from bone marrow lack expression of CD44 protein

Despite significant progress in our understanding of mesenchymal stem cell (MSC) biology during recent years, much of the information is based on experiments using in vitro culture-selected stromal progenitor cells. Therefore, the natural cellular identity of MSCs remains poorly defined. Numerous studies have reported that CD44 expression is one of the characteristics of MSCs in both humans and mice; however, we here have prospectively isolated bone marrow stromal cell subsets from both human and mouse bone marrow by flow cytometry and characterized them by gene expression analysis and function assays. Our data provide functional and molecular evidence suggesting that primary mesenchymal stem and progenitor cells of bone marrow reside in the CD44(-) cell fraction in both mice and humans. The finding that these CD44(-) cells acquire CD44 expression after in vitro culture provides an explanation for the previous misconceptions concerning CD44 expression on MSCs. In addition, the other previous reported MSC markers, including CD73, CD146, CD271, and CD106/VCAM1, are also differentially expressed on those two cell types. Our microarray data revealed a distinct gene expression profile of the freshly isolated CD44(-) cells and the cultured MSCs generated from these cells. Thus, we conclude that bone marrow MSCs physiologically lack expression of CD44, highlighting the natural phenotype of MSCs and opening new possibilities to prospectively isolate MSCs from the bone marrow.     

Substance P stimulates the recovery of bone marrow after the irradiation

The therapeutic use of ionizing radiation (e.g., X-rays and γ-rays) needs to inflict minimal damage on non-target tissue. Recent studies have shown that substance P (SP) mediates multiple activities in various cell types, including cell proliferation, anti-apoptotic responses, and inflammatory processes. The present study investigated the effects of SP on γ-irradiated bone marrow stem cells (BMSCs). In mouse bone marrow extracts, SP prolonged activation of Erk1/2 and enhanced Bcl-2 expression, but attenuated the activation of apoptotic molecules (e.g., p38 and cleaved caspase-3) and down-regulated Bax. We also observed that SP-decreased apoptotic cell death and stimulated cell proliferation in γ-irradiated mouse bone marrow tissues through TUNEL assay and PCNA analysis. To determine how SP affects bone marrow stem cell populations, mouse bone marrow cells were isolated and colony-forming unit (CFU) of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) was estimated. SP-pretreated ones showed higher CFUs of MSC and HSC than untreated ones. Furthermore, when SP was pretreated in cultured human MSC, it significantly decreased apoptotic cells at 48 and 72 h after γ-irradiation. Compared with untreated cells, SP-treated human MSCs showed reduced cleavage of apoptotic molecules such as caspase-8, -9, -3, and poly ADP-ribose polymerase (PARP). Thus, our results suggest that SP alleviates γ-radiation-induced damage to mouse BMSCs and human MSCs via regulation of the apoptotic pathway.     

Osmotic response of oocytes using a microscope diffusion chamber: a preliminary study comparing murine and human ova

The hydraulic conductivity (Lp) of the plasma membrane determines how cells respond to the stresses of dehydration encountered during cryopreservation. We have used a microscope diffusion chamber which allows for direct real-time observation of the dynamic osmotic response of individual cells in microvolume suspension to compare the Lp of murine and human unfertilized ova. In this system, the response of an individual cell to the induced osmotic imbalance is documented via a series of photomicrographs or videotape; from these data the Lp can be computed. Donated human preovulatory oocytes were compared with macroscopically normal human ova, 43 hr after insemination, which had failed to fertilize (Ff) and with murine ova collected 13 hr post human chorionic gonadotropin injection. The permeability coefficients were 0.65 +/- 0.43, 0.84 +/- 0.39, and 0.36 +/- 0.07 micron3/micron2/atm/min. The results suggest that it may be possible to use Ff ova for experiments to design suitable cryopreservation procedures.     

Enrichment isolation of adipose-derived stem/stromal cells from the liquid portion of liposuction aspirates with the use of an adherent column

Background aimsAdipose-derived stem/progenitor cells (ASCs) are typically obtained from the lipoaspirates; however, a smaller number of ASCs can be isolated without enzymatic digestion from the infranatant liposuction aspirate fluid (LAF). We evaluated the effectiveness of an adherent column, currently used to isolate mesenchymal stromal cells from bone marrow, to isolate LAF cells.MethodsWe applied peripheral blood (PB), PB mixed with cultured ASCs (PB-ASC), and LAF solution to the column and divided it into two fractions, the adherent (positive) and the non-adherent (negative) fractions. We compared this method with hypotonic hemolysis (lysis) for the red blood cell count, nucleated cells count and cell compositions as well as functional properties of isolated mesenchymal cells.ResultsThe column effectively removed red blood cells, though the removal efficiency was slightly inferior to hemolysis. After column processing of PB-ASC, 60.5% of ASCs (53.2% by lysis) were selectively collected in the positive fraction, and the negative fraction contained almost no ASCs. After processing of LAF solution, nucleated cell yields were comparable between the column and hemolysis; however, subsequent adherent culture indicated that a higher average ASC yield was obtained from the column-positive samples than from the lysis samples, suggesting that the column method may be superior to hemolysis for obtaining viable ASCs. Mesenchymal differentiation and network formation assays showed no statistical differences in ASC functions between the lysis and column-positive samples.ConclusionsOur results suggest that a column with non-woven rayon and polyethylene fabrics is useful for isolating stromal vascular fraction cells from LAF solutions for clinical applications.     

Isolation and Characterization of Multipotential Mesenchymal Cells from the Mouse Synovium

The human synovium contains mesenchymal stem cells (MSCs), which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs) have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.     

Preparation of lipid-free human hemoglobin by dialysis and ultrafiltration

Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid-free hemoglobin. High pressure liquid chromatography analysis of lipid-free hemoglobin (LFHB) indicates 99-100% protein purity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119-139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one-half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.     

Do microRNAs regulate bone marrow stem cell niche physiology?

The adult bone marrow, situated within the bone cavity, comprises three distinct stem cell populations: hematopoietic stem cells (HSCs), mesenchymal stromal/stem cells (MSCs) and endothelial progenitor/stem cells (EPCs). HSCs are a well-characterized population of self-renewing cells that give rise to all blood cells. The definition of MSCs is more complex due to the limited understanding of MSC properties. In general, MSCs are considered multipotent stromal cells that are able to differentiate into various cell types, including osteoblasts, chondrocytes and adipocytes. Compared to HSCs and MSCs, EPCs are a newly discovered population of stem/progenitor cells with the capacity to differentiate into endothelial cells, the cells forming the inner lining of a blood vessel.     

Expression, regulation and function of AC133, a putative cell surface marker of primitive human haematopoietic cells

To explore the physiological significance of AC133 expression on human haematopoietic cells, we phenotyped normal and malignant human haematopoietic cells for AC133 expression, evaluated the utility of AC133 for isolating human stem/progenitor cells in comparison to other known early haematopoietic cell markers, investigated the role of AC133 in regulating hematopoiesis, and evaluated the possibility that MYB might regulate AC133. We found that while human CD34+ progenitor cells expressed AC133, expression was rapidly downregulated during differentiation. In apparent contrast, AC133 mRNA was detectable in cells isolated from CFU-Mix, BFU-E, CFU-GM and CFU-Meg colonies. Human cord blood CD34+ cells expressed AC133 at higher levels than their normal bone marrow counterparts. In apparent contrast to normal primitive haematopoietic cells, the AC133 protein was undetectable on cells from 24 different human haematopoietic cells lines, even though the majority of these cells expressed AC133 mRNA. Since CD34, AC133 and the c-kit (KIT) receptor are all co-expressed on human stem/progenitor cells, we compared the ability of monoclonal antibodies directed against each of these proteins to isolate early progenitor cells. Using these antibodies and magnetized particles in a standard immunoaffinity isolation protocol, we found that anti-CD34 and anti-KIT MoAbs could isolate > 80-90% of the clonogeneic cell population present in a given marrow sample. Anti-AC133 MoAbs recovered approximately 75-80% of CFU-GM and CFU-Meg, but only about 30% of CFU-Mix and BFU-E. Perturbation of AC133 expression with antisense oligodeoxynucleotides (AS ODN) resulted in transient downregulation of AC133 protein on human CD34+ cells but no apparent effect on cell survival or cloning efficiency ex vivo. Finally, downregulation of MYB expression with AS ODN had no effect on the AC133 expression at either the mRNA or protein level. Based on these results, we conclude that AC133 offers no distinct advantage over CD34 or c-kit as a target for immunoaffinity based isolation of primitive hematopoietic cells, that AC133 expression is not required for normal hematopoietic progenitor cell development in vitro, and finally that AC133 expression may not be MYB-dependent.     

Human CD133-derived bone marrow stromal cells establish ectopic hematopoietic microenvironments in immunodeficient mice

Cultured adherent bone marrow stromal cells (BMSCs) are capable of forming ectopic hematopoietic microenvironments (HMEs) in immunodeficient mice. However, the cell surface phenotype of the native bone marrow stem/progenitor cell that gives rise to BMSCs that support hematopoiesis remains poorly defined. We recently reported the derivation of human BMSC-like cells (CD133BMSCs) by magnetic cell sorting against Prominin-1 (CD133), an epitope expressed by embryonic, fetal, and adult stem cells. Here we demonstrate that CD133BMSCs are capable of forming ectopic HMEs. Cultured adherent CD133BMSCs derived from sorted CD133-positive cells lacked CD133 expression, but were uniformly positive for CD146, an epitope recently described to identify self-renewing osteoprogenitor cells that could transfer the HME. CD133BMSCs were genetically-tagged by lentivirus, expanded, and seeded into HA/TCP/fibrin constructs that were implanted subcutaneously. After 60 days, CD133BMSCs produced human osteocytes, osteoblasts, adipocytes, and reticular cells that supported murine hematopoiesis. CD133BMSCs that were not transduced with lentivirus also formed HMEs. Control constructs seeded with human dermal fibroblasts formed connective tissue, but failed to form HMEs. Our data indicate that CD133 expression identifies a native human bone marrow stem/progenitor cell that gives rise to BMSCs capable of forming the HME.     

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3(-/-)) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3(-/-) mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3(-/-) mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3(-/-) mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.     

Membrane trafficking and osmotically induced volume changes in guard cells

Guard cells rapidly adjust their plasma membrane surface area while responding to osmotically induced volume changes. Previous studies have shown that this process is associated with membrane internalization and remobilization. To investigate how guard cells maintain membrane integrity during rapid volume changes, the effects of two membrane trafficking inhibitors on the response of intact guard cells of Vicia faba to osmotic treatments were studied. Using confocal microscopy and epidermal peels, the relationship between the area of a medial paradermal guard-cell section and guard-cell volume was determined. This allowed estimates of guard-cell volume to be made from single paradermal confocal images, and therefore allowed rapid determination of volume as cells responded to osmotic treatments. Volume changes in control cells showed exponential kinetics, and it was possible to calculate an apparent value for guard-cell hydraulic conductivity from these kinetics. Wortmannin and cytochalasin D inhibited the rate of volume loss following a 0-1.5 MPa osmotic treatment. Cytochalasin D also inhibited volume increases following a change from 1.5 MPa to 0 MPa, but wortmannin had no effect. Previous studies showing that treatment with arabinanase inhibits changes in guard-cell volume in response to osmotic treatments were confirmed. However, pressure volume curves show that the effects of arabinanase and the cytochalasin D were not due to changes in cell wall elasticity. It is suggested that arabinanase, cytochalasin D, and wortmannin cause reductions in the hydraulic conductivity of the plasma membrane, possibly via gating of aquaporins. A possible role for aquaporins in co-ordinating volume changes with membrane trafficking is discussed.     

人造血干/祖细胞多态性的研究:Ⅲ.骨髓CD34~ 造血干/祖细胞功能亚群的分析

CD 34~ 造血干/祖细胞(HSC/HPC)是十分异质性的,由多个不同功能亚群所构成,在分化方向与重建造血等方面差异显著。本文对正常人骨髓CD 34~ HSC/HPC各亚群进行了较全面的分析。首先以阳性选择策略,采用Isolex~(TM) 50免疫磁球分选术富集骨髓CD 34~ HSC/HPC,其纯度>90%,随之采用免疫荧光抗体双标记二维流式细胞仪对其测定,发现高纯度的CD 34~ HSC/HPC可分为八个不同亚群:1.CD 34~ /CD 71~-(23.4%—56.6%)与CD 34~ /CD 71~ (33.4%—66.6%);2.CD34~ /CD 45~-(80.8%—82.5%)与CD34~ /CD 45~ (8.1%—11.2%);3.CD 34~ /CD 33~-(20.4%—80.6%)与CD 34~ /CD 33~ (14.6%—64.8%);4.CD 34~ /DR~-(6.3%—11.0%)与CD 34~ /DR~ (82.8%—85.5%)。用免疫胶体金——免疫桥酶联组化双染色对上述亚群进一步分析的结果与流式细胞仪的高度一致,为研究各亚群的功能与生物学特性提供了坚实基础     

Impact of parathyroid hormone on bone marrow-derived stem cell mobilization and migration

Parathyroid hormone (PTH) is well-known as the principal regulator of calcium homeostasis in the human body and controls bone metabolism via actions on the survival and activation of osteoblasts. The intermittent administration of PTH has been shown to stimulate bone production in mice and men and therefore PTH administration has been recently approved for the treatment of osteoporosis. Besides to its physiological role in bone remodelling PTH has been demonstrated to influence and expand the bone marrow stem cell niche where hematopoietic stem cells, capable of both self-renewal and differentiation, reside. Moreover, intermittent PTH treatment is capable to induce mobilization of progenitor cells from the bone marrow into the bloodstream. This novel function of PTH on modulating the activity of the stem cell niche in the bone marrow as well as on mobilization and regeneration of bone marrow-derived stem cells offers new therapeutic options in bone marrow and stem cell transplantation as well as in the field of ischemic disorders.     

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

Stabilins are expressed in bone marrow sinusoidal endothelial cells and mediate scavenging and cell adhesive functions

Bone marrow sinusoidal endothelial cells have a specific function as a site of transmigration of hematopoietic stem and progenitor cells and mature blood cells between bone marrow and blood stream. However, the specific characteristics of bone marrow sinusoidal endothelial cells are still largely unclear. We here report that these cells express stabilin-1 and stabilin-2, which in liver sinusoidal endothelial cells have been identified as endocytic scavenger receptors for several ligands, including SPARC and hyaluronan. We show here that intravenously injected formaldehyde-treated serum albumin, advanced glycation end-products, and collagen I α-chains were taken up by bone marrow sinusoidal endothelial cells, showing that these cells have a scavenging function and thereby may modulate bone marrow vascular stem cell niches. Importantly, we show hyaluronan mediated adhesion of hematopoietic stem and progenitor cells to stabilin-2-transfected cells, suggesting that stabilin-2 contributes to adhesion and homing of circulating stem and progenitor cells to bone marrow.