Human bone marrow colony growth in agar-gel

A technique for growing human bone marrow cell colonies in agar-gel medium is described. “Feeder layers” containing 1 × 10⁶ normal human peripheral white blood cells are used as the stimulus for colony growth. Human bone marrow aspirates are collected in heparinized syringes and plated as 2 × 10⁵ cells on “feeder layers.” Normal human bone marrow yields 32–102 colonies per 2 × 10⁵ cells plated. Colonies are almost exclusively granulocytic. Growth rate of colonies is slower than with mouse bone marrow but colonies reach a comparable size (500–1500 cells) at days 12–16.     

Clonogenic stromal cell count in the bone marrow of mice

The number of fibroblast colonies in bone marrow cultures depends on FCFC concentration in explanted cells and FCFC cloning efficiency. For mouse bone marrow the efficiency of fibroblast colony formation increases in the presence of the feeder (irradiated bone marrow of spleen cells). Colony-stimulating feeder activity does not depend on the presence of phagocytic and stromal cells in the feeder cell population. Trypsinization of the bone marrow leads to the release of additional FCFC and the increase of their concentration in bone marrow cell suspensions.     

The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.     

The non-equivalence of mouse and human marrow culture in the assay of granulopoietic stimulatory factors

Mouse bone marrow forms colonies of granulocytes and monocytic phagocytes when cultured in the presence of human plasma, urine or “feeder layers” prepared from human leukocytes. By contrast, human marrow produces colonies in the presence of leukocyte feeder layers but not in the presence of plasma or urine. It has been tacitly assumed that the response of mouse marrow to human blood leukocyte feeder layers is a measure of physiological substances released by those leukocytes which might control human granulopoiesis. This assumption however, has never been put to the test by comparing the response of mouse and human marrow to stimulation by leukocytes from the same individual. This has been done in the present study by using leukocytes from normal and leukemic subjects. Different human marrows responded similarly to stimulation by the same normal feeder layers, but there was no quantitative or qualitative correlation between the response of human and mouse marrows. Feeder layers from patients with acute granulocytic leukemia did not stimulate colony growth in normal human marrow but were as potent in stimulating mouse marrow colony growth as were feeder layers of normal leukocytes. We conclude that different factors may stimulate human and mouse marrows and that assays of granulopoietic factors of human origin should in future be carried out in human rather than mouse marrows.     

Effects of B-lymphocytes from different organs on hemopoietic colony formation in the spleen by bone marrow cells]

The influence of B-lymphocytes from various sources on splenic colony formation was studied in the syngeneic system. B-lymphocytes were obtained by panning with IgG-fraction of rabbit anti-mouse Ig, absorbed on Petri dishes. In addition, adherent cells, Thy-1+ and SC-1+ were eliminated from the fraction of Ig(+)-cells. SC-1- and SC-1+ fractions, containing, respectively, stem cells and T-lymphocyte precursors, were obtained by panning with IgG-fraction of rabbit anti-SC-1 serum. SC-1- cells transferred to irradiated syngeneic mice did not induce colony formation in the spleen. Introduction of SC-1- and SC-1+ cells induced formation of colonies. A similar helper effect occurred when SC-1(-)-cells were introduced with bone marrow or lymph node B-cells, but not with splenic B-cells. Splenic, but not bone marrow and lymph node B-cells inhibited colony formation by combination of SC-1- and SC-1+ cells. All effects of Ig+ cells were abolished by treatment of cells with rabbit anti-MBLA serum. Thus, B-cells of various origin can either enhance or inhibit colony formation. The enhancing of inhibitory effect after B (MBLA+)-cells elimination from suspension of bone marrow and lymph node (but not spleen) Ig(+)-cells resulted from the activity of B-contrasuppressors.     

Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts

The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.     

差速贴壁分离法:获得小鼠骨髓内皮祖细胞诱导分化培养的最佳贴壁时间

本文旨在比较差速贴壁方法分离的不同时间点贴壁的小鼠骨髓单个核细胞诱导分化为内皮祖细胞的生物学特性,探讨最适宜贴壁时间.Ficoll密度梯度离心分离小鼠骨髓单个核细胞,接种于预先铺有纤维连接蛋白的培养板上,定义为1d贴壁细胞组,取1d非贴壁细胞再接种为3d贴壁细胞组,继续培养2d取非贴壁细胞再接种为3d非贴壁细胞组,继续...     

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.     

细脚拟青霉田间分离菌株间的异核现象

本文报道细脚拟青霉(paecilomyces tenuipes)不同田间分离菌株单孢子后代间的异核现象。用来自荣园、菜地、水稻田三种生境的四个菌株(803、2801、1401和3101)的单孢子培养后代,在加有酵母膏和麦芽糖的改良萨氏培养基上进行配接实验,仅在803与1401两菌株间能形成异核体,其频率为13.5%。在配接实验中两亲和菌落交界处长出白色致密的菌丝簇组成的实线可推测为异核体。从来自菌丝簇组线的每个单菌丝尖端培养物中分离出30个以上的单孢子,井分别移接到 PDA 平板上。将来源于单孢子的菌落与两亲本菌落进行比较,有三个单菌丝尖端培养物(C_3、B_3、F_3)重现了两亲本类型或出现了新的菌落类型,从而异核现象得到证实。     

The continued requirement for inducer for the development of macrophage and granulocyte colonies

Rat bone marrow cells were seeded as mass cultures or for cloning together with inducer required for colony formation, and at various times after seeding, the cells were re-seeded for cloning either with or without inducer. The results indicate that the development of both macrophage (M) and granulocyte (G) colonies requires a continued supply of the inducer. No M or G colonies were produced when the inducer was replaced by erythropoietin.     

鼠骨髓间充质干细胞的分离培养及定向诱导分化为内皮样细胞

目的：探讨体外大鼠骨髓间充质干细胞（rBMMSCs）的分离培养和血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）对其定向诱导为内皮样细胞（ELCs）的可行性。方法：采用Percoll（1.073g/ml）分离液分离骨髓单个核细胞,用含10%胎牛血清（FBS）的LG-DMEM培养基贴壁纯化培养,倒置显微镜、免疫细胞化学法、流式细胞仪、MTT法、透射电镜（TEM）联合对rBMMSCs形态、表型、生长曲线、细胞周期以及超微结构进行鉴定;诱导后的细胞,采用倒置显微镜观察细胞形态,免疫细胞化学法检测CD31、CD144（VE-cadherin）和CD34表达以及摄取Dil-ac-LDL、结合FITC-UEA-1的功能特点。结果：rBMMSCs呈长梭形,漩涡状排列。细胞生长曲线显示潜伏期、对数生长期和平台期,符合干细胞的生长规律。透射电镜结果表明：rBMMSCs有两种不同的形态结构,其中体积较小、核质比大、胞质内细胞器稀少者为处于未分化或分化较低状态的幼稚型rBMMSCs。细胞周期分析显示：第4代细胞G0/G1期为95.67%,表明绝大部分细胞处于非增殖状态;诱导后的部分细胞形态可见类似ELCs改变,表达血管内皮细胞（ECs）特异性表面标志CD31、CD34和CD144,具有摄取Dil-ac-LDL以及结合FITC-UEA-1的功能特点。结论：采用Percoll密度梯度离心与贴壁培养相结合的方法所培养的rBMMSCs在体外具有定向诱导分化为ELCs的潜能,可能成为血管组织工程理想的种子细胞来源。     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Response of female Lutzomyia longipalpis to host odour kairomones from human skin

Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) is the vector of Leishmania chagasi, the aetiologic agent of visceral leishmaniasis in the New World. In the present study, the response of female sandflies from Jacobina, Brazil, to human odours from six different volunteers was investigated. Glass Petri dishes were handled by different volunteers and then exposed to female sandflies. There was a significant difference between subjects in that some individuals were more attractive or less repellent to sandflies. Response of flies to handled Petri dishes was higher during the first minutes of observation, suggesting the presence of volatile compounds in hand odours. Extracts of glass Petri dishes that had been handled by the volunteers were made with organic solvents such as acetone, methanol, pentane and ether. These were then concentrated and tested for sandfly response. Only extracts carried out with non-polar solvents such as pentane and ether were able to transfer odours from handled glass Petri dishes onto clean dishes. The attractivity of male and female human subjects was monitored for 80 days, and minor fluctuations in attractiveness were observed.     

Sonic Hedgehog Signaling Pathway Supports Cancer Cell Growth during Cancer Radiotherapy

Tumor growth after radiotherapy is a commonly recognized cause of therapeutic failure. In this way, we examined tumor cell growth after radiotherapy by establishing a cancer cell growth model in vitro. We accomplished this model by seeding non-irradiated firefly luciferase2 and green fluorescent protein fusion gene (Fluc) labeled living cancer reporter cells onto a feeder layer of irradiated cancer cells. The living tumor cell growth was monitored by bioluminescence imaging. The living reporter cells grew faster when seeded onto lethally irradiated feeder cells than when seeded onto non-irradiated feeder cells or when seeded in the absence of feeder cells. We found that the expression levels of the Shh and Gli1 proteins, both of which are critical proteins in Sonic hedgehog (SHH) signaling, were increased after irradiation and that this expression was positively correlated with reporter cell growth. Moreover, the dying cell stimulation of living tumor cell growth was enhanced by the addition of SHH signaling agonists and inhibited by SHH signaling antagonists. SHH agonists also enhanced reporter cell growth in the absence of irradiated feeder cells, suggesting this mechanism plays a role in feeder cell growth stimulation. Given these results, we conclude that SHH signaling activation plays an important role during tumor repopulation after radiotherapy.     

BACTERIA COLONY IDENTIFICATION USING VIDEO IMAGING

This paper describes an image analysis method to detect and locate Salmonella colonies grown on Petri dishes, on two different media. The colonies are described using their common feature characteristics: black center surrounded by a clear halation. Morphological image analysis functions are preferred to gray levels because of the color inhomogeneity of the background even under optimal lighting. The results obtained show the good ability of this method for detecting and locating but further work is needed to validate it on a greater number of Petri dishes.     

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.     

A method for detecting restriction endonucleases in bacterial colonies

A simple technique is proposed for detection of bacterial restriction endonucleases. Analysis is performed directly in the cells from colonies cultivated on Petri dishes. The cells collected with an inoculation loop are treated with lysozyme and Triton X-100. After centrifugation the supernatant is tested for endonuclease activity. The technique enables up to 100 colonies to be tested for 3-4 h.     

CFU-F from dog marrow: a colony assay and its significance

A colony assay method is described for studying dog fibroblast colony development in marrow cells derived from resected ribs. The assay showed an increased number of fibroblast colony forming units (CFU-F) in cell suspensions prepared from resected ribs compared to cell suspensions prepared from bone marrow aspirates or from peripheral blood. A linear relationship between the number of cells plated and the number of fibroblastoid colonies was demonstrated in each case. The proportion of phagocytic cells was lower in cultures prepared from resected ribs than in those prepared from bone marrow aspirates. Staining for acid phosphatase and with sudan black showed differences between phagocytic cells and non-phagocytic fibroblasts. When left in plastic dishes for 2 hrs, 81% +/- 10% of the CFU-F adhered to the plastic dishes. Velocity sedimentation separation showed a modal sedimentation rate of 6.49 mm/h.     

Differences in the in vitro growth pattern of fresh and cryopreserved granulo-monopoietic precursors

A study of the in vitro growth model of human granulo-monopoietic precursors (CFU-GM) before and after cryopreservation using both leukocyte feeder layers and GCT conditioned medium as the source of colony stimulating activity (CSA) is reported. The number of colonies produced with fresh cells was linearly related to the amount of marrow seeded with both CSA sources, whereas after cryopreservation this was true with feeder layers, and with GCT only at relatively high cell concentrations. This might indicate the production of granulopoietic stimulators on the part of a second population that is at least partly resistant to freezing. It seems more likely, however, that these results depend mainly on a sublethal damage to CFU-GM induced by freezing, thus making the cells hyporesponsive to some forms of CSA, such as those contained in GCT conditioned medium.