Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung

Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well-differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies. Disclosure of potential conflicts of interest is found at the end of this article.     

Adipocyte differentiation of human marrow mesenchymal stem cells reduces the supporting capacity for hematopoietic progenitors but not for severe combined immunodeficiency repopulating cells

Bone marrow stromal cells provide a microenvironment for hematopoiesis. Adipocytes are the major stromal cell phenotype in bone marrow, but their function in hematopoiesis is poorly understood. In this study, we compared the hematopoietic-supporting capacity of adipocytes and their progenitor, mesenchymal stem cells (MSCs), by culturing human cord blood (CB) CD34+CD38- hematopoietic progenitor cells (HPCs) on a layer of adipocytes or MSCs. CB CD34+CD38- cells cultured on MSCs generated higher proportions of CD34+CD38- HPCs and colony-forming cells than those cultures on a layer of adipocytes, indicating an inferior hematopoietic support by adipocytes. However, CB CD34+CD38- HPCs cultured on MSCs and adipocytes were equally capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings show that differentiation of MSCs into adipocytes is accompanied by the loss of capacity to support mature HPCs, but not transplantable SCID-repopulating cells.     

Bmp2 is required for migration but not for induction of neural crest cells in the mouse.

Bone morphogenetic protein (BMP) signaling is essential for neural crest development in several vertebrates. Genetic experiments in the mouse have shown that Bmp2 is essential for the genesis of migratory neural crest cells. Using several markers and a transgenic reporter approach, we now show that neural crest cells are induced in Bmp2 null mutant embryos, but that these cells fail to migrate out of the neural tube. The absence of migratory neural crest cells in these mutants is not due to their elimination by cell death. The neuroectoderm of Bmp2-/- embryos fail to close and create abnormal folds both along the anterior-posterior and medio-lateral axes, which are associated with an apparent medio-lateral expansion of the neural tube. Finally, our data suggest that the molecular cascade downstream of BMP signaling in early neural crest development may be different in mouse and avian embryos.     

Oral naloxone reduces constipation but not antinociception from oral morphine in the rat.

Oral administration of naloxone (10 mg/kg) antagonized the slowing of the intestinal transit caused by oral morphine (1, 2.5 and 5 mg/kg) in rats. Oral administration of naloxone (10 mg/kg) did not prevent the antinociceptive effect of orally administered morphine (2.5 mg/kg) in the tail-flick test carried out on rats. It is concluded that oral naloxone locally blocks the constipating effect of morphine, while it fails to reduce the central action of morphine due to extensive metabolization after oral administration.     

Telomerase activity in hybrids between telomerase-negative and telomerase-positive immortal human cells is repressed in the different complementation groups but not in the same complementation group of immortality

The expression of telomerase is essential for cells to be immortalized, and most immortal cell lines possessed telomerase activity. Using the cell fusion technique, it has been shown that mortal and telomerase-negative phenotypes of normal cells are dominant over immortal and telomerase-positive phenotypes, suggesting that the normal cells possessed dominant repressor-type activity for telomerase expression. Several telomerase-negative immortal human cell lines were reported, in which telomerase-independent mechanisms was supposed to maintain telomere length. We aimed at seeing whether the telomerase-negative phenotype of these immortal cells is dominant over telomerase-positive phenotype of other immortal cells in correlation with cellular mortality. Results showed that, when telomerase-positive and -negative immortal parental cell lines belonging to the different complementation groups were fused, telomerase-negative mortal hybrid clones arose, i.e. telomerase-negative phenotype was dominant as well as mortal phenotype. However, when immortal hybrid cells arose from telomerase-positive and -negative immortal parents belonging to either the same or different complementation groups, they were all telomerase-positive, i.e. telomerase-negative phenotype appeared to be recessive. Telomerase-negative immortal hybrid was never established from any combinations between telomerase-negative and -positive immortal parental cells.     

L-selectin expression differentiates T cells isolated from different lymphoid tissues in cattle but does not correlate with memory.

L-selectin (LECAM-1, LAM-1) was expressed by a high proportion of CD4+ and CD8+ T cells, as well as almost all of the gamma delta T-cell receptor (TcR)+ (WC1+) T cells, isolated from blood, lymph nodes or tonsils. CD4+ T cells in the lamina propria of the gut villi and CD8+ T cells in the villous epithelium as well as the majority of WC1+ T cells in the gut mucosa were L-selectin-. The proportion of T cells from Peyer's patches that synthesized the molecule was intermediate between the value for blood and gut mucosa. Expression of L-selectin therefore marks T cells in cattle with a distinct tissue distribution that correlates with its function as the peripheral node homing receptor. The proportion of CD4+ and CD8+ T cells in the circulation that were L-selectin+ decreased with age. Unlike CD45R, expression of L-selectin was not related to CD4 T-cell memory as judged by proliferation in transformation assays to soluble antigen. Three-colour immunofluorescent staining demonstrated four subpopulations of CD4 and CD8 T lymphocytes in peripheral blood mononuclear cells (PBMC) that were CD45R+, L-selectin+; CD45R+, L-selectin-; CD45R-, L-selectin+; CD45R-, L-selectin-. CD4(4) memory cells were CD45R- and L-selectin+ or L-selectin-. Taken with earlier studies the reported observations demonstrated that only one of the four phenotypes of the CD4+ T cells in blood is present in the lamina propria of the gut villi and these are CD45R-, L-selectin-. Two of the four phenotypes of CD8+ T cells were present in the gut epithelium; these were CD45R+, L-selectin- or CD45R-, L-selectin-. Expression of the bovine molecule was not rapidly down-regulated on T cells following activation by exposure to phorbol myristate acetate.     

Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo

Notch receptors and their ligands contribute to many developmental systems, but it is not apparent how they function after birth, as their null mutants develop severe defects during embryogenesis. Here we used the Cre-loxP system to delete the Delta-like 1 gene (Dll1) after birth and demonstrated the complete disappearance of splenic marginal zone B cells in Dll1-null mice. In contrast, T cell development was unaffected. These results demonstrated that Dll1 was dispensable as a ligand for Notch1 at the branch point of T cell-B cell development but was essential for the generation of marginal zone B cells. Thus, Notch signaling is essential for lymphocyte development in vivo, but there is a redundancy of Notch-Notch ligand signaling that can drive T cell development within the thymus.     

Loss of NF1 allele in Schwann cells but not in fibroblasts derived from an NF1-associated neurofibroma.

Neurofibromas, the hallmark of neurofibromatosis 1, are composed mainly of Schwann cells and fibroblasts. Inactivation of both NF1 alleles is the cause of these benign tumors, but it is unknown which cell type is the progenitor. In this study, we selectively cultured Schwann cells from an NF1-associated neurofibroma. Fibroblasts were also obtained by culturing the tumor cells under standard conditions. Using four intragenic markers, we genotyped the NF1 locus in the original tumor and in the derived Schwann cells and fibroblasts. Loss of heterozygosity for two informative markers, which indicates loss of one NF1 allele, was found in Schwann cells but not in fibroblasts. This result suggests that genetic alterations of the NF1 gene in Schwann cells are responsible for the development of neurofibromas.     

X inactivation patterns of closely, but not distantly, related cells are highly correlated: little evidence for stem cell plasticity in normal females

The early, random nature of X inactivation should cause related cells to have similar, but distinctive, active X chromosomes. We assessed the frequency of stem cell plasticity using X inactivation proportions (XIPs), determined at the human androgen receptor locus, in paired tissue samples from healthy individuals. Tissues sampled were stomach (n = 18 informative females), duodenum (n = 18), colon (n = 10) with corresponding peripheral blood samples (n = 33), and varicose veins (n = 28) with corresponding T cells (n = 26) and peripheral blood granulocytes (n = 25). XIPs from samples thought to have common stem cell origins were highly correlated: multiple samples from single vein, r = .80 (n = 24); T cells versus granulocytes, r = .67 (n = 23); duodenum versus stomach, r = .63 (n = 12). Blood cells and vessels are derived from a common hemangioblast, but XIP correlations were moderate or poor: vein versus T cells, r = .42 (n = 26); vein versus granulocytes, r = .11 (n = 25). X inactivation is believed to be a late process in gut, especially hind-gut, with corresponding independence from blood precursors. Correlations with blood cells were low: stomach, r = .23 (18); duodenum, r = .21 (18); colon, r = .034 (10). Any crossover of stem cells between different organs during adult life should increase correlations with age; no such increase was seen. This study confirms that XIPs can be used to track stem cell populations, provides a theoretical basis for the power of such studies, and indicates that hemopoietic stem cell plasticity is, at most, uncommon in normal humans.     

Activation via the antigen receptor is impaired in T cells,but not in B cells from patients with common variable immunodeficiency

The patients included in this study belong to a subset of common variabie immunodeficiency (CVID) patients whose peripheral blood T cells have a T cell receptor (TCR)-mediated activation defect leading to impaired expression of the interleukin (IL)-2 gene upon stimulation with recall antigens (tetanus toxoid, Escherichia coli) or superantigens (staphylococcal enterotoxins). In the present report we demonstrate that the patients' peripheral blood T cells failed to generate the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) following stimulation with superantigen or mAb specific for the monomorphic region of the TCR β-chain. Patients' T cell lines were also impaired in generating Ins(1,4,5)P₃ when stimulated with tetanus toxoid-pulsed autologous monocytes. Addition of a second or third co-stimulatory signal provided by recombinant IL-2, CD28 or both had no effect on the Ins(1,4,5)P₃ formation of the patients' antigen-driven T cell lines. The T cell activation defect, however, was not absolute, as Ins(1,4,5)P₃ formation in the patients' T cells after phytohemagglutinin or aluminium fluoride stimulation was normal. The impairment in signal transduction via the T cell antigen receptor was limited to the patients' T cells, as no activation defect after ligation of surface immunoglobulin, the antigen receptor on B cells, could be detected.     

An amino acid change in the exodomain of the E2 protein of Sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells.

In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV(STD)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV(LM17), did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SV(LM17) in greater detail. In yield assays, the titer of SV(LM17) produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SV(STD), in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SV(LM17) was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SV(LM17), viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SV(LM17)-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SV(LM17) was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype.     

Fatty acids are important for the Frank-Starling mechanism and Gregg effect but not for catecholamine response in isolated rat hearts

In some pathophysiological conditions myocardial metabolism can switch from mainly long chain fatty acid (LCFA) oxidation to mainly glucose oxidation. Whether the predominant fatty acid or glucose oxidation affects cardiac performance has not been defined. In a buffer perfused isovolumetrically contracting rat heart, oxidation of endogenous pool LCFA was avoided by inhibiting carnitine-palmitoyl-transferase I (CPT-I) with oxfenicine (2 mM). In order to restore fatty acid oxidation, hexanoate (1 mM), which bypasses CPT-I inhibition, was added to the perfusate. Three groups of hearts were subjected to either an increase in left ventricular volume (VV, +25%) or an increase in coronary flow (CF, +50%), or inotropic stimulation with isoproterenol (10(-8) and 10(-6) m). The increase in VV (the Frank-Starling mechanism) increased rate-pressure product (RPP) by 21 +/- 2% under control conditions, but only by 6 +/- 2% during oxfenicine-induced CPT-I inhibition. The contractile response to changes in VV recovered after the addition of hexanoate. Similar results were obtained in hearts, in which an increase in CF was elicited (the Gregg phenomenon). Isoproterenol caused a similar increase in contractility regardless of the presence of oxfenicine or hexanoate. In all groups, a commensurate increase in oxygen consumption accompanied the increase in contractility. The fatty acid oxidation is necessary for an adequate contractile response of the isolated heart to increased pre-load or flow, whereas the inotropic response to adrenergic beta-receptor stimulation is insensitive to changes in substrate availability.     

Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body.

Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.     

CD4(+) T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.

The role of CD4(+) lymphocytes in the establishment of lentivirus infection in macrophages has been studied in an in vivo system of lentivirus infection where CD4(+) lymphocytes are not the targets for infection. Using the non-T-cell-tropic lentivirus, maedi-visna virus (MVV), in CD4-depleted sheep, we have found that CD4(+) T cells were required for MVV infection in macrophages but not dendritic cells. CD4-depleted sheep had significantly lower levels of MVV-infected cells in lymph nodes and efferent lymph after MVV challenge in the drainage area of the lymph node. Due to the absence of virus in combination with the lack of CD4(+) T helper cells, virus-specific immune responses were reduced. There was delayed induction of cytotoxic T cell precursors, a marked reduction in virus-specific in vitro proliferative responses, and a delay in the appearance of MVV-specific antibodies. By contrast, CD4 depletion had no effect on the establishment of MVV infection in afferent lymph dendritic cells migrating from the skin infection site to the lymph node.     

Deletion of self-reactive T cells in the donor-derived T cells but not in the host-derived T cells in fully allogeneic radiation chimeras. Mls-reactive T cells in allogeneic radiation chimeras

Kinetics of T cells bearing V beta 6 capable of recognizing Mls-1a were examined in the thymus and peripheral lymphoid organs of two allogeneic bone marrow chimeras; AKR/J(H-2k, Thy1.1,Mls-1a)----C3H/He(H-2k, Thy1.2,Mls-1b) and AKR/J----C57BL/6(H-2b,Thy1.2, Mls-1b). Sequential appearance of host- and donor-derived T cells occurred in the thymus and the peripheral lymphoid organs of both AKR----C3H and AKR----B6 chimeras. The first cells to repopulate the thymus were Thy1.2+ host-derived radioresistant cells, which were synchronized in their development. The host-derived cells in thymus of AKR----B6 chimeras differentiate more rapidly than those in AKR----C3H chimeras. An almost complete replacement from host-derived cells to donor-derived cells occurred by day 21 after reconstitution in AKR----C3H and AKR----B6 chimeras. In the donor-derived thymocytes, none of CD4- or CD8-single positive thymocytes expressed high density of V beta 6 in either AKR----C3H or AKR----B6 chimeras, whereas the host-derived thymocytes in AKR----B6 chimeras contained an appreciable number of CD4-single positive thymocytes bearing V beta 6. In the peripheral lymphoid organs, T cells bearing V beta 6 were virtually abolished in Thy1.1+ cell pool of both AKR----C3H and AKR----B6 chimeras. While V beta 6+ T cells of host-origin were detected in the peripheral lymphoid organs in AKR----B6 chimeras. These result indicated that the donor-derived mature T cells showed deletion of V beta 6 in the thymus and the peripheral lymphoid organs in both AKR----C3H and AKR----B6 chimeras, whereas lack of V beta 6 deletion was observed in the host-derived mature T cells in the AKR----B6 chimeras. These results suggested that the host-derived thymocytes may likely to escape undergoing a negative selection against donor-phenotype in the radiation bone marrow chimeras.     

Crescentic glomerulonephritis in CD4- and CD8-deficient mice. Requirement for CD4 but not CD8 cells.

The contribution of CD4 and CD8 cells to crescentic glomerulonephritis (GN) was studied in mice genetically deficient in CD4, CD8, and with combined CD4 and CD8 (CD4/CD8) deficiency. Wild-type (C57BL/6) mice developed GN with mild proliferative changes 7 days after an intravenous dose of sheep anti-mouse glomerular basement membrane globulin. Crescents were observed in 12.5 +/- 6.1% of glomeruli on day 14. On day 21, 51.5 +/- 7.3% of glomeruli were affected by crescents, and mice had marked azotemia and proteinuria. CD4 and combined CD4/CD8-deficient mice developed minimal evidence of GN. On day 21, their glomeruli showed only mild proliferative changes and crescents, azotemia, and proteinuria were absent. In contrast, CD8-deficient mice developed severe crescentic GN with three of five mice dying on day 20 with ascites and edema. The two mice surviving to day 21 had severe azotemia. Crescent development was accelerated (day 14, 51.6 +/- 2.4% of glomeruli; day 20 or 21, 62.0 +/- 4.0% of glomeruli). These studies demonstrate that CD4 cells are crucial for the development of crescentic GN in mice and that genetic absence of CD8 cells accelerates disease. They support the hypothesis that crescent formation is a manifestation of CD4-dependent (and CD8-independent) delayed type hypersensitivity in the glomerulus.     

CD4+ T cells, but not CD8+ T cells, are required for the development of experimental autoimmune gastritis.

Murine autoimmune gastritis, induced by neonatal thymectomy, is characterized by a mononuclear infiltrate within the gastric mucosa, loss of parietal and zymogenic cells and circulating autoantibodies to the gastric H/K ATPase. The infiltrate contains both CD4+ and CD8+ T cells. Here we have investigated the roles of CD4+ and CD8+ T cells in the development of gastritis by in vivo treatment with depleting rat anti-CD4 and anti-CD8 monoclonal antibodies. Depletion of CD4+ T cells decreased the incidence of gastric mononuclear infiltrates from 63% (5/8), observed in normal rat immunoglobulin G (IgG)-injected mice, to 8% (1/12) and also abolished the production of antigastric autoantibodies. In contrast, depletion of CD8+ T cells did not reduce the incidence of gastritis. The absence of CD8+ T cells in the infiltrate of the stomach of anti-CD8(+)-treated mice was confirmed by immunocytochemistry. These results argue that neonatal thymectomy-induced autoimmune gastritis is mediated by CD4+ T cells and that CD8+ T cells do not play a significant role in the development of the gastric lesion.