Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep

We previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human fetal liver hematopoietic stem cells in sheep after transplantation in utero. That the engraftment of long-term repopulating pluripotent stem cells occurred in these animals was shown here by the fact that transplantation of human CD45+ cells isolated from bone marrow of these chimeric animals into preimmune fetal sheep resulted in engraftment and expression of human cells. Marrow cells were obtained from three chimeric sheep at 3.2-3.6 yr after transplant. The relative percentage of human CD45+ cells present in these marrows was 3.3 +/- 0.32%. A total of 29 x 10(6) CD45+ cells were isolated by panning, pooled, and transplanted into six preimmune sheep fetuses (4.8 x 10(6) cells/fetus). All six recipients were born alive. Hematopoietic progenitors exhibiting human karyotype were detected in marrows of two lambs soon after birth. Cells expressing human CD45 antigen were also detected in blood and marrow of both lambs. Human cell expression has been multilineage and has persisted for > 1 yr. These results demonstrate that the expression of human cells in this large animal model resulted from engraftment of long-term repopulating pluripotent human stem cells.     

In utero partial liver resection in the rabbit model: a study on fetal tissue regeneration

This study compared step-back preparations in curved canals of resin blocks using nickel-titanium K-files and stainless steel K-files. Forty canals in resin blocks were cross-sectioned at 3 levels: 1 to 2 mm from the apical foramen, middle of the curve, and coronal. Direct digital computer images were recorded before and after instrumentation. Superimposition of the images combined with digital subtraction computer software allowed direct measurement of area instrumented, distance of transportation, and shape analysis. Time for instrumentation was recorded. Results showed Ni-Ti files to cause significantly less transportation and remain more centered at the apical level (p < 0.05). Area removed by instrumentation was significantly greater for stainless steel files at the middle level (p < 0.05). Cross-sectional shape of the instrumented canal was not significantly different (p < 0.05). It took significantly longer to prepare a canal with Ni-Ti K-files in resin blocks compared to stainless steel (p < 0.05). Resin simulated canals showed similar results compared to canals in extracted roots using an identical methodology.     

Regulation of tissue plasminogen activator production in cultured human fetal mesangial cells

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.     

Identification of bipotential progenitor cells in human liver development

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.     

Techniques for studying stimulation of fetal hemoglobin production in human erythroid cultures

This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with beta hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with beta hemoglobinopathies.     

Expression of genes for human globin in the cell line K-562: an experimental model for the study of fetal erythropoiesis

Human leukemia K-562(S) cells are a useful model system to study the relationship between cell proliferation and induced erythroid differentiation. In these studies K-562(S) cells were cultured in alpha -medium, 10% fetal calf serum and induced to express erythroid genes by 75 microM hemin, 1.2 mM butyric acid or 1.5 ng/ml actinomycin D. Cell number was determined using a ZF Coulter Counter and the increase in the proportion of hemoglobin-containing cells was detected by a specific colorimetric reaction with benzidine. The characterization of the synthesized hemoglobins was performed by cellulose-acetate gel electrophoresis of post-mitochondrial supernatants. By cloning K-562(S) cells in semi-solid medium (O,33% agar) containing 75 microM hemin a variant cell line, denominated K-562(S6), have been isolated which does not undergo terminal cell division but does express human globin genes and accumulates on the average 12 pg of Hb/cell. K-562(S6) cells accumulate, upon exposure to 75 microM hemin, mostly Hb Gower 1 (zeta 2 epsilon 2) and low amounts of Hb X (epsilon 2 gamma 2) and Hb Portland (zeta 2 gamma 2), being suitable for studies focused on the expression of embryonic globin genes and on the molecular mechanisms controlling the switching from embryonic-type to fetal-type hemoglobin accumulation, when in the human embryo zeta and epsilon globin genes become less active, being sharply increased accumulation of alpha and gamma globin chains.     

Asymmetric cell divisions sustain long-term hematopoiesis from single-sorted human fetal liver cells

Curve fitting procedures for bioanalytical assays are based on classical linear least squares (LSE) theory. A common procedure is to select among various models and weighting factors using the R2 as a goodness-of-fit criterion. It is questionable whether R2 is the most appropriate criterion for model selection. This is compounded by an often subjective removal of outliers. In this article, statistical curve fitting and diagnostic criteria are proposed. The fitting procedure is a Box-Cox-type power transformation of the data. The optimal transformation is obtained as the one that minimises the sum of squared deviations. Potential outlying standards are screened during the diagnostics stage as those whose jackknife percent deviations exceed 20%. The main advantage of this method is that it is objective and uniformly applicable across analytical techniques. Furthermore, the optimal transformation obtained in this way is unique. The results are demonstrated by comparing the power model to the R2 approach through the statistical analysis of 2094 analytical batches for 91 projects using various analytical techniques, namely GC, HPLC, LCMS and GCMS. The results indicate that the power model is robust and that QC batch acceptance using the power model is at least as good as the current method. These results hold true across all analytical techniques. It is thus strongly suggested that curve fitting and standard outlier detection for bioanalytical assays should be based on a power model and on jackknife percent deviations method with acceptable cut-off values.     

Immunophenotypic characterization of human fetal liver hematopoietic stem cells during the midtrimester of gestation

OBJECTIVE: Our purpose was to define the extent to which gestational age influences the number of fetal liver cells that coexpress phenotypic markers associated with hematopoietic stem cells and major histocompatibility antigens. STUDY DESIGN: Fetal liver cells from abortuses of 9 to 24 weeks of gestation were studied (n = 61). Low-density nucleated liver cells were isolated on a discontinuous density gradient and subsequently incubated with antibodies that recognize markers of hematopoietic stem cells (i.e., CD33, CD34, CDw90, CD117, and CD123). Human leukocyte antigen class I (A, B, C) and class II (DR) antigens were also determined on these cells. Each sample was analyzed by immunocytochemistry and flow cytometry. Analysis of variance was used for statistical analysis. RESULTS: Of the markers measured, only the percentage of CD123-positive cells increased significantly with gestational age (p < 0.01). The percentage of triple-positive cells (CD34+, CD117+, and CD123+) increased with age but did not reach significance (p = 0.05). Human leukocyte A, B, and C antigens were expressed on all nucleated cells from 9 to 24 weeks of gestation. Human leukocyte DR antigen, however, was expressed only on 50% of these cells. The percentage of cells that expressed both hematopoietic stem cell markers and DR antigen did not vary with gestational age. CONCLUSIONS: From 9 to 24 weeks of gestation the number of human fetal liver hematopoietic stem cells that coexpress major histocompatibility antigens increases with advancing gestational age, largely because the percentage of these cells remains constant while the liver mass increases.     

Transplantation of human fetal brain cells: theological-ethical aspects

Using fetal brain cells for therapeutic purposes after a spontaneous abortion can be justified under similar pre-conditions like the removal of an organ post mortem. With an artificially induced abortion, however, there is the danger of justifying the act of abortion by combining it with that positive purpose. In this case any therapeutic utilization of fetal brain cells would only be justifiable, if every step of such a procedure could completely be isolated from the ethically unjustifiable abortion.     

Human neonatal ovary: proposal of a three-dimensional model

A model of human neonatal ovary is presented, derived from morphometric, evaluations carried out on left ovaries removed from five full-term neonates with a 46, XX karyotype, free from malformations of the genital apparatus. According to this model, the gonad can be represented by a triaxial ellipsoid with a central medullary core surrounded by a cortical stratum of constant thickness. The germinal population, consisting of follicles and primitive cortical tissue, occupies the cortex, intermingled with the interstitium or stroma. In the cortex it is then possible to describe an outer layer formed by primitive cortical tissue, and an inner portion occupied by follicles. The primary and secondary follicles fill the portion near the medulla and the primordial ones are contained in the middle and outer zones. Since the variability observed among ovaries is slight, we can propose a mean model of neonatal ovary in which the spatial relationships among the different components, the total number of follicles and their position in the cortex can be calculated.     

Colloidal silica-coated tissue culture dishes for primary cell cultures: growth of rabbit renal proximal tubule cells

The use of colloidal silica as a substratum for primary cultures of differentiated cells has significant advantages over classic tissue culture polystyrene. In this report, the growth and the level of expression of differentiated function of primary rabbit renal proximal tubule (RPT) cell cultures on colloidal silica is examined, using hormonally defined serum-free medium. Primary RPT cells grew to confluence more rapidly on colloidal silica than on tissue culture polystyrene (TC+). Moreover, following three passages, the RPT cells increased in number threefold more than parallel cultures on TC+. The morphology of primary RPT cells on colloidal silica were found by means of transmission electron microscopy to possess a polarized morphology with a brush border, and differentiated markers were retained even after passaging, including the Na+/glucose cotransport system and Glut 7.     

The influence of steroids on erythropoiesis in mouse fetal liver cell cultures: relevance of cell cycle state

It has been shown that erythropoietin-mediated stimulation of heme synthesis in mouse fetal liver cells in vitro is correlated with hydroxyurea sensitivity. Assuming that OHU is specifically cytotoxic for cells in DNA synthesis this suggests that erythropoietin sensitivity may be related to this phase of the cell cycle. The direct effects of 19-nortestosterone and 3beta-hydroxy-5beta-pregnan-20-one on heme synthesis correlated with the capacity of these steroids to initiate DNA synthesis. It is suggested that the ability of these steroids to increase the proportion of cells in the Ep-sensitive phase of the cell cycle is probably the mechanism responsible for the erythropoietic effects of these agents. 17beta-hydroxy-5alpha-androstan-3-one appears to have a different mode of action since it has only minimal effects on heme synthesis and did not increase hydroxyurea sensitivity.     

In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.     

Endothelial cells in culture: an experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [3H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor-alpha and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selection and vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also in rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.     

Transcatheter liver lobar ablation: an experimental trial in an animal model

Complete embolization of tumor tissue together with surrounding liver sufficiently prevents collateral blood supply to the tumor, offering curative treatment for hepatic malignancies. The present experiment was designed to test the feasibility of hepatic lobar ablation by means of the transcatheter chemoembolization technique. Five groups of rats (n = 6) were treated with a mixture of iodized oil/ethanol in ratios of 5:1, 4:1, 3:1, 1:1, and 1:0, which was injected selectively into the right-lobe artery until saturation during open surgery. Another group (n = 6) was studied using in vivo microscopy to observe the distribution of the mixture in the liver and changes in hepatic microcirculation. Ethiodol/ethanol mixture entered the portal vein after injection into the hepatic artery creating dual, complete arterial and portal venous embolization. Lobar ablation effects were achieved in 2 weeks in the 5:1, 4:1, and 3:1 ratio groups, indicated by the lobe/liver weight measurements (p < 0.001 vs normal liver). Hepatic arterial administration of the Ethiodol/ethanol mixture creates dual hepatic arterial and portal venous embolization, achieving a lobar ablation effect.     

Effect of some preservatives on pregnancy and fetal development in experimental animals

70 rats in 7 groups of 10 rats each were studied to determine the effects of an oral administration of 3.3-dibromo-5.5-dichloro-2.2-diphenyl-methane during the period of organogenesis (the 6th-15th day of pregnancy). 5 groups received a dose of 1/1000, 1/500, 1/250, 1/100, and 1/50 of the preparation (LD50) at 1 ml/100/gm of weight. 1 control group received olive oil in a dose of 1 ml/100 gm of weight and the other was given thalidomide at 1/500 LD50. The animals were killed on the 20th day of pregnancy and the fetuses given gross inspections regarding the number of living and dead, the weight of the fetus and of the placenta, the length of the fetus and its tail, and the sex. A statistical analysis using the student t test with a probability of 5% was performed on these data. Autopsies and macroscopic investigations of the liver and kidneys were performed. The development of occipital and interparietal bones, ossification centers in the sternum, and the number of ribs were also assessed. This preparation did not exert any inhibiting influence on fetal development or cause an increase in fetal mortality.