Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

Relationship between tooth and long bone size

Canonical correlations between tooth and long bone dimensions showed a greater correlation for Anglo-Saxons and apes than for Nineteenth Century Londoners, i.e., coefficients of 0.75 for gorilla, 0.72 for chimpanzee, 0.69 for orang-utans, 0.74 for Anglo-Saxons, but 0.53 for Nineteenth Century Londoners. Although based upon limited sample sizes and limited metrical profiles of teeth and long bones, the data support the thesis that modern Europeans are under reduced selection pressure to maintain tooth size compared with apes or ancient man.     

Immunologic unresponsiveness of mouse spleen sensitized to allogenic tumors.

CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is $\text{1}\text{1}\text{2}\text{–2}\text{1}\text{2}$ months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.     

How do trabeculae affect the calculation of structural properties of bone?

One difficulty that arises in an analysis of the cross-sectional properties of bone is whether to include cancellous bone in the analysis. The purpose of this paper is to determine how different amounts of cancellous bone affect the measurement of structural properties of bone cross-sections. Thirty-two tibial and femoral cross-sections were chosen at random from a series of cross-sectioned nonhuman primate bones. Geometrical properties were calculated for the cross-sections, and torsional and bending stress analyses were performed. The results suggest that the effect of including cancellous bone in the analysis is closely related to the amount of bone, where it lies within the cross-section, and the type of analysis performed. Including cancellous bone in calculations of structural properties of bone cross-sections may cause the strength and stiffness of the bone to be exaggerated.     

鳅鮀鱼类鳔囊结构及系统发育研究(鲤形目:鲤科)

本文应用分支系统学原理对鳅(鱼它)鱼类的鳔囊、鳔及相关结构进行了特征分圻,并以(鱼句)亚科作为外类群。解剖结果表明,鳅(鱼它)鱼类的鳔分四种类型;分析结果进一步证实鳅(鱼它)鱼类是一个单源群,作为一亚科可分为异鳔鳅(鱼它)属和鳅(鱼它)属,后者又分为原鳅(鱼它)和鳅(鱼它)二个亚属。     

造血细胞活力冷冻损伤的可恢复性

人骨髓冻存后其造血祖细胞活力有一定程度下降,本研究对这种下降的可逆性作了初步观察。结果发现,用双层法和单层法作CFU-GM培养时,未冻存骨髓集落产率相近,冻存骨髓双层法的CFU-GM产率高于单层法。骨髓细胞用20％FM-CM、PHA-LYCM、PHA-PMCM预孵育2h后,分别测定其CFU-GM、BFU-E与CFU-Mix,发现这种孵育过程对未冻存骨髓的集落产率无明显影响,而冻存骨髓的集落产率在孵育后可升高(GEMmeg除外)。说明骨髓造血祖细胞对冻存的损伤反应不均一,部分受损细胞在一定条件下可以恢复其增殖活力。这对于用冻存骨髓作骨髓移植可能有一定意义。     

Long-term expression of gene introduction into normal human T-lymphocytes by retroviral-mediated gene transfer

Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.