Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.

We have generated immunodeficient scid-/scid- (SCID)-transgenic mice expressing the genes for human interleukin 3, granulocyte/macrophage-colony stimulating factor, and stem cell factor. We have compared engraftment and differentiation of human hematopoietic cells in transgenic SCID mice with two strains of nontransgenic SCID mice. Human bone marrow cells carrying the CD34 antigen or human umbilical cord blood were injected into sublethally irradiated recipients. Human DNA was detected by polymerase chain reaction in peripheral blood and bone marrow of 14 of 28 transgenic SCID mice after transplantation, but in only 2 of 15 nontransgenic SCID littermates at a 10-fold lower level. Bone marrow cultures 8 wk after transplantation of cord blood gave rise to human burst-forming unit erythroid, colony-forming unit granulocyte/macrophage, or granulocyte/erythroid/macrophage/megakaryocyte colonies. Engraftment was observed for up to 6 mo in transgenic SCID mice, twice as long as nontransgenic littermates or previous studies in which transplanted SCID mice were given daily injections of growth factors. We conclude that the level and duration of engraftment of human cells in SCID mice can be improved by expression of human cytokine transgenes and that transgenic SCID mice are an efficient model system for the study of human hematopoiesis.

Main branch stent deformation following difficult side branch rewiring and balloon dilatation. Rare complication of provisional T stenting.

Coronary artery bifurcations are one of the largest challenges in interventional cardiology. Presented is the case of a patient in whom restenosis of a drug-eluting stent (DES) occurred as a consequence of guide wire re-crossing between the main vessel stent struts and the vessel wall in the proximal part of DES, and consequential balloon crushing of the proximal portion of the DES. Initially, the complication was not recognized because of a good angiographic result and absence of intravascular ultrasound (IVUS) guidance during the procedure. During the second procedure, IVUS analysis explained the mechanism of the DES failure. The problem was solved with the implantation of a new DES.

Fetal rat intestinal absorption of horseradish peroxidase from swallowed amniotic fluid.

Horseradish peroxidase (HRP) injected into amniotic fluid is swallowed by rat fetuses and within 3-6 h reaches the gut lumen. This macromolecular protein is then absorbed by the columnar lining cells via a system of apical cytoplasmic tubules formed by invaginations of the plasma membrane. From cytoplasm subjacent to the brush border HRP is transported, within vacuoles, to the supranuclear region, where some is retained for at least 18 h, and to interepithelial spaces. Extracellular enzyme is then found throughout the epithelial basement membrane and between connective tissue cells of the mucosal and submucosal layers Finally, HRP can be detected within lumina of blood and lymphatic capillaries, strongly suggesting that it is transported from the intestine to the circulation.

Protein absorption by the intestine of the fetal rat in utero.

Horseradish peroxidase (molecular weight, about 40,000) injected into the amniotic sacs in pregnant rats has been identified ultrastructurally, 6 to 18 hours later, within the fetal intestine in the absorptive cells and the underlying vascular endothelium. This indicates that macromolecular protein within amniotic fluid swallowed by the fetus can be absorbed and transported by fetal intestine, and may indicate that physiological compounds can be transported by this enteric route to contribute to fetal development.

Retrovirus receptor mRNA expression correlates with gene transfer efficiency in pluripotent hematopoietic stem cells.

Hematopoietic stem cells (HSC) from bone marrow, peripheral blood and cord blood are important in clinical transplantation. However, their use in gene therapy protocols is still limited by a low level of transduction efficiency. In addition to the cell cycling block to retrovirus transduction, we recently demonstrated that the low level of retrovirus receptor mRNA in mouse HSC correlated with the low level of amphotropic retrovirus transduction in these cells. Similarly, we found low levels of mRNA encoding the amphotropic retrovirus receptor in human bone marrow Lin CD34+ CD38- HSC. In an effort to identify an alternative population of human HSC that might be more efficiently transduced, we assayed HSC populations from cord blood for mRNA encoding the amphotropic retrovirus receptor. High levels of receptor mRNA were present in HSC from previously cryopreserved cord blood compared with HSC from fresh bone marrow and fresh cord blood. The HSC from cryopreserved cord blood are excellent candidates for gene therapy protocols.

Increased 2,5-adenylate synthetase activity in the spleens of BALB/c mice during hypoxia-stimulated erythropoiesis.

In response to prolonged, intermittent exposure to hypoxia, the spleens of adult BALB/c mice displayed an initial increase and subsequent decrease in erythropoietic activity. The enzyme 2,5-adenylate synthetase was assayed during this period, and a direct relationship was found between the rate of red cell production and enzyme activity; that is, 2,5-adenylate synthetase activity was maximum in the spleen cell populations that contained the largest number of nucleated erythroid cells and minimum in those populations that contained the fewest nucleated erythroid cells. In contrast to this finding, synthetase activity was inversely related to the number of lymphocytes present in these spleen cell populations. On the basis of these observations, it appears that 2,5-adenylate synthetase is present in nucleated erythroid cells. If active in late erythroblasts, 2,5-adenylate synthetase may function as an inhibitor of DNA synthesis and/or hemoglobin synthesis.

Pluripotent hematopoietic stem cells of low and high density can repopulate W/Wv mice.

We have studied several features of pluripotent hematopoietic stem cells (PHSC) and day-12 spleen colony-forming units (CFU-S12) in murine bone marrow. C57BL/6J marrow cell suspensions were separated by elutriation and fractions were obtained at flow rates (FR) of 25 ml/min, 29/30 ml/min, 35 ml/min, and with the rotor off. All four fractions contained PHSC that could repopulate W/Wv mice, but significant numbers of CFU-S12 were found only in the three higher FR fractions. Cells in the FR29/30 fraction were shown to have almost three-fold more repopulating activity than fresh marrow in a competitive repopulation assay. The PHSC in fractions separated by elutriation were enriched by depleting cells expressing specific lineage markers with monoclonal antibodies and magnetic immunobeads. As few as 10(4) lineage negative (lin-) cells from FR35 or 10(5) lin--cells from FR25 conferred long-term multilineage repopulation in W/Wv mice, as demonstrated by Southern blot analysis of DNA from recipient thymus and bone marrow. We conclude that PHSC are heterogeneous for cell size and density and that the highest concentration of PHSC resides in the subset of intermediate density present in the FR29/30 fraction.

Electron microscopy of human fetal erythroid cells before and after cryopreservation.

Fetal erythropoiesis was studied in human livers at 10 to 12 weeks of gestation. The most primitive blood cells were often observed in large indentations of the surface of hepatocytes and the plasma membranes of the two cell types were adherent at sites of attachment. Erythroid cell maturation occurred predominantly in the lumen of the sinusoids. Cell suspensions obtained from fetal livers were centrifuged, frozen at -196 degrees C, thawed and studied by electron microscopy. The primitive cells were morphologically altered by these procedures. Changes included damage to mitochondria and cell membranes and vacuole formation. Erythroblasts, by comparison, were virtually intact and even displayed some indications of reestablished functions within 10 minutes after thawing.

Inhibition of CFU-E in rat bone marrow perfused with 2'-5'-oligoadenylate core.

Several 2'-5' oligoadenylate (2-5A) core molecules were tested for their inhibitory effect on erythroid colony-forming units (CFU-E). These compounds were introduced into the CFU-E microenvironment by vascular perfusion of isolated rat hind limbs. The marrow microvasculature did not appear to be damaged by these procedures when examined by electron microscopy. After a brief in situ exposure to 2-5A core, the marrow cells were grown in methylcellulose medium with added erythropoietin (Ep). Maximum percent inhibition of CFU-E was obtained with 2-5A trimer core. The tetramer core was less effective, and no inhibition of colony formation was seen in cultures of marrow perfused with 2-5A dimer core. From this it was concluded that the inhibitory effect of 2-5A trimer core was highly specific. Furthermore, this effect was of long duration, since when 2-5A trimer core was given just prior to Ep the CFU-E were blocked in their differentiative response to Ep for 48-72 h.

Interleukin-7R alpha mRNA expression increases as stem cells differentiate into T and B lymphocyte progenitors.

The gamma common (gamma c) chain is a partner in several interleukin receptor complexes, including the interleukin-2 receptor (IL-2R), IL-4R, and IL-7R. Mutations in the gamma c gene are associated with X-linked severe combined immunodeficiency (SCID). Using reverse transcriptase-PCR, we examined the level of mRNA-encoding gamma c and its partners in mouse pluripotent hematopoietic stem cells (PHSCs), which repopulate both bone marrow and thymus. We also assayed developing lymphocytes to define which, if any, IL-R complexes are expressed at the earliest stage of T and B lymphocyte maturation. RNA extracted from bone marrow-derived PHSCs did not contain detectable levels of mRNA-encoding IL-7R alpha. However, the most primitive (CD4- CD8-) T cells from the thymus and the most primitive (c-kit+ B220+) B cells from bone marrow contained high levels of IL-7R alpha mRNA. There were no detectable differences between PHSCs and primitive or more mature T and B cells for expression of gamma c mRNA. We conclude that the onset of IL-7R formation occurs at the earliest stage of differentiation of T and B lymphocytes. Our findings are consistent with the hypothesis that the absence of an intact IL-7R (IL-7R alpha and gamma c) may be a critical loss that interrupts lymphopoiesis.

The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

Increased erythropoiesis and 2'5'-A polymerase activity in the marrow and spleen of phenylhydrazine-injected rats.

The rate of CFUE production in adult rats varies considerably according to whether the animals are rendered anemic by short or long term treatment with phenylhydrazine. The bone marrow responds during 2 months of phenylhydrazine injections with a progressive increase in CFUE, the spleen shows a rapid and larger rise after 3 daily injections followed by a decline to near zero, control numbers, after prolonged administration of the drug, and the liver never develops erythropoietic activity. Endogenous colonies, that is, colonies that grow in plasma clots without added erythropoietin, are most numerous in bone marrow after phenylhydrazine treatment, are always few in the spleen and are never present in cultures of liver cells. In both marrow and spleen, there is a direct correlation between the rate of erythropoiesis and 2'5'-A polymerase activity. These findings suggest that production of the enzyme may be an early event in erythroid cell differentiation.

Granulocyte colony-stimulating factor mobilized peripheral blood stem cells enter into G1 of the cell cycle and express higher levels of amphotropic retrovirus receptor mRNA.

We compared the cell cycle status and expression of mRNA for the amphotropic retroviral receptor in hematopoietic stem cells isolated from bone marrow and cytokine mobilized peripheral blood. CD34+ cells from six normal volunteers were enriched by immune selection from steady-state bone marrow and granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (10 microg/kg/day for 5 days). Cell cycle status of the phenotypically primitive CD34+CD38- hematopoietic stem cell population was analyzed using a four-color flow cytometry technique that distinguished the G0, G1, and S/IG2/M phases of the cell cycle. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to measure mRNA expression of the amphotropic retroviral receptor. Peripheral blood hematopoietic stem cells had 2.6-fold more cells in the G1 phase of the cell cycle compared to steady-state bone marrow. Furthermore, lineage CD34+CD38- cells from G-CSF mobilized peripheral blood had a fourfold higher level of amphotropic retrovirus receptor mRNA. In conclusion, we found that CD34+ CD38- hematopoietic stem cells isolated from G-CSF mobilized peripheral blood differ from those isolated from steady-state bone marrow in that a significant proportion have entered the G1 phase of the cell cycle and express higher levels of amphotropic receptor mRNA. These biologic properties are consistent with the reported rapid recovery of hematopoietic function following transplantation with peripheral blood hematopoietic stem cells and make these cells a preferred target for retroviral-based gene transfer.

Alternative viral envelopes for oncoretroviruses to increase gene transfer into hematopoietic stem cells.

The hematopoietic stem cell is the target for gene therapy of human blood disease. Low retroviral receptors for the commonly used vectors and quiescence of hematopoietic stem cells are believed to be major obstacles to the success of gene therapy. The development of new stem cell assays has allowed better understanding of the biology and phenotype of hematopoietic stem cells, leading to selection of highly enriched populations of hematopoietic stem cells. Quantitation of retrovirus receptors on these enriched populations of hematopoietic stem cells has resulted in the identification of subpopulations of cells expressing high levels of retrovirus receptors. New promising retrovirus envelopes are being developed. In this review, we discuss those issues that may help to resolve the problem of low gene transfer efficiency into human hematopoietic stem cells.

Sustained hypertransfusion and induction of a transplantable myeloid leukemia in RLV-A-infected BALB/c mice.

Infection of BALB/c mice with the RLV-A virus typically results in an erythropoietic dysplasia characterized by hepatosplenomegaly, erythroblastosis, erythroblastemia and severe anemia without reticulocytosis. Mice hypertransfused weekly with 75%-packed red cells for 42 days prior to RLV-A infection and viral potency controls manifested this typical RLV-A response. Mice that were hypertransfused prior to and following RLV-A infection never developed the "typical" RLV-A pathogenesis. Instead, a transplantable myeloid leukemia was established. Although the reason for altered pathogenesis remains uncertain, it seems plausible that continued hypertransfusion, presumably after establishment of an altered granulopoietic microenvironment, resulted in a completely different viral expression and development of the transplantable myeloid leukemia.

Molecular mechanism for the inhibitory action of interferon on hematopoiesis.

Spleen cell suspensions obtained from adult mice were separated by Ficoll/Hypaque and Percoll density gradient centrifugation. The enriched erythroblast populations were maintained in liquid culture medium for 8 hours with 10,000 units of murine interferon (IFN) alpha and beta. Exposure of these cell cultures to murine IFN alpha and beta resulted in a 48% to 70% increase in 2-5adenylate synthetase (2-5AS) activity. In parallel studies, adult mice were injected daily for 1 or 2 weeks with recombinant human IFN alpha A/D (rHuIFN alpha A/D) at a dose of 10(6) or 10(7) units/kg body weight. This treatment did not significantly affect body weight but did produce a mean 70% increase in spleen wet weight and a mean 46% increase in number of nucleated cells per spleen. This increase in number of splenic hematopoietic cells did not result in a corresponding increase in number of circulating cells. In fact, during this 1 to 2 week period the hematocrit dropped from 45% to 38% in mice injected with high dose rHuIFN alpha A/D. From these findings we propose that IFN induces an early 2-5AS activity in erythroblasts and megakaryocytes. This 2-5AS activity, which is known to inhibit protein synthesis in other cell systems, is thought to be responsible for the block or prolongation in blood cell maturation observed in the present studies.

Transplanted adult bone marrow cells repair myocardial infarcts in mice.

Occlusion of the anterior descending left coronary artery leads to ischemia, infarction, and loss of function in the left ventricle. We have studied the repair of infarcted myocardium in mice using highly enriched stem/progenitor cells from adult bone marrow. The left coronary artery was ligated and 5 hours later Lin- c-kit+ bone marrow cells obtained from transgenic male mice expressing enhanced green fluorescent protein (EGFP) were injected into the healthy myocardium adjacent to the site of the infarct. After 9 days the damaged hearts were examined for regenerating myocardium. A band of new myocardium was observed in 12 surviving mice. The developing myocytes were small and resembled fetal and neonatal myocytes. They were positive for EGFP, Y chromosome, and several myocyte-specific proteins including cardiac myosin, and the transcription factors GATA-4, MEF2, and Csx/Nkx2.5. The cells were also positive for connexin 43, a gap junction/intercalated disc component indicating the onset of intercellular communication. Myocyte proliferation was demonstrated by incorporation of BrdU into the DNA of dividing cells and by the presence of the cell cycle-associated protein K167 in their nuclei. Neo-vascularization was also observed in regenerating myocardium. Endothelial and smooth muscle cells in developing capillaries and small arterioles were EGFP-positive. These cells were positive for Factor VIII and alpha smooth muscle actin, respectively. No myocardial regeneration was observed in damaged hearts transplanted with Lin- c-kit- bone marrow cells, which lack bone marrow-regenerating activity. Functional competence of the repaired left ventricle was improved for several hemodynamic parameters. These in vivo findings demonstrate the capacity of highly enriched Lin- c-kit+ adult bone marrow cells to acutely regenerate functional myocardium within an infarcted region.

Amphotropic retrovirus transduction of hematopoietic stem cells.

Mice treated with cytokines for 5 days have large numbers of hematopoietic stem cells (HSCs) in their peripheral blood and bone marrow at 1 and 14 days after the last injection. We fractionated the HSCs from the bone marrow of these mice using elutriation at flow rates of 25, 30 and 35 ml/min. The subpopulations of HSCs from cytokine-treated mice show a 3- to 8-fold higher level of mRNA encoding the amphotropic retrovirus receptor (amphoR) compared with the corresponding HSC subpopulation from untreated mouse bone marrow. In an earlier study with mouse HSCs we showed a direct correlation between high levels of amphoR mRNA and efficient retrovirus transduction. We have now utilized our gene transfer protocol to assay amphotropic retrovirus transduction efficiency using HSCs from the bone marrow of mice treated with granulocyte-colony stimulating factor/stem cell factor (G-CSF/SCF). To extend these findings to a more clinically relevant protocol we analyzed the amphoR mRNA levels in HSCs from human cord blood and adult bone marrow. The amphoR mRNA level in HSCs from human bone marrow and fresh cord blood was detectable at an extremely low level compared with the HSC population in cryopreserved cord blood samples. The 12- to 22-fold increase in amphoR mRNA in HSCs from cryopreserved cord blood renders these HSCs likely candidates for high efficiency, gene transfer.