Culture of colony-forming hematopoietic progenitor cells from human peripheral blood

Summary Methods for culturing hematopoietic colonies from human peripheral blood are described in this report. Granulocyte-macrophage progenitor cells (CFU-GM: colony forming units-granulocyte/macrophage) are grown in 35-mm dishes containing 4 ml of 0.35% agarose in Iscove's modified Dulbecco's medium (IMDM) with 20% prescreened fetal bovine serum (FBS). Colony-stimulating activity is provided by leukocyte-conditioned medium, and nonactivated autologous T lymphocytes or recombinant granulocyte- and granulocyte/macrophage-colony stimulating factor or both. This procedure minimize the growth inhibition caused by monocytes. Erythroid progenitor cells (BFUe: burst forming units-erythroid) are cultured in 1 ml of 0.35% agarose in IMDM with 30% FBS and conditioned medium from human bladder carcinoma cell line 5637 or recombinant interleukin 3. Both assays have proved useful in studying the enrichment of peripheral blood hematopoietic cells.     

Identification of a novel bovine serum protein which is involved in human T-cell leukemia virus type I (HTLV-I)-induced syncytium formation

Summary  By immunizing rats with cocultured HTLV-I-positive ILT8M2 and HTLV-I-negative MOLT-4 cells, we isolated a monoclonal antibody (mAb), designated as mAb R21, which enhances the syncytium formation induced by coculturing ILT8M2 cells with MOLT-4 cells. The antigen recognized by mAb R21 was found on the surface of all T-cell, fibroblastoid, and epithelial cell lines, and a part of B-cell and myelomonocytoid cell lines. MAb R21 reacted with an approximately 17-kDa protein from ILT8M2 and MOLT-4 cell lysates in both nonreducing and reducing conditions by immunoblotting. Immunoprecipitation experiments using surface-labeled cells revealed that a 17-kDa protein is present on the surface of both ILT8M2 and MOLT-4 cells. Since the enhancing activity by mAb R21 of syncytium formation was observed only in the presence of a factor contained in fetal calf serum (FCS) which seems to bind to mAb R21, we purified this serum factor from FCS using a mAb R21- coupled Sepharose 4B column. The purified protein, designated as R21 protein, was revealed to be O-glycosylated but not N-glycosylated protein of approximately 17 kDa. The partial amino acid sequence of this protein indicates that R21 protein is a novel bovine serum protein which has approximately 90% amino acid homology with bovine platelet factor 4, a member of CXC chemokine family. These results indicate that the R21 protein on the surface of cells and/or in FCS may play an important role in the process of HTLV-I-induced syncytium formation by as yet unknown mechanism. Received July 6, 1998 Accepted August 29, 1998     

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.     

Reproducible proliferative responses of salamander (Ambystoma mexicanum) lymphocytes cultured with mitogens in serum-free medium.

There are several reports that proliferative responses (tritiated thymidine incorportation (^3HTdR)) of salamander splenocytes cultured with phytohemagglutinin-P (PHA) or concanavalin A (Con A) in 1% fetal bovine serum (FBS)-supplemented medium are either statistically insignificant or never approach the magnitude typically observed in similarly treated cultures of frog lymphocytes. The present study confirms these findings, but also reports highly significant and reproducible PHA-induced proliferation of axolotl splenocytes and thymocytes when the medium is supplemented with 0.25% bovine serum albumin (BSA) rather than 1% FBS. In one study, splenocytes from six of six axolotls cultured in BSA-supplemented medium displayed a dose-dependent response to PHA with stimulation indices (SLs) ranging from 4.2 to 14.1. In contrast, SLs of PHA-treated cells from the same animals, cultured in parallel in FBS-supplemented medium, ranged from 0.8 to 3.0. In a kinetic study (cells harvested from days 3–7), maximal proliferation in BSA-supplemented medium was noted after 5 days; cells cultured in parallel in FBS-containing medium were not responsive to the mitogen at any time point. Although axolotl splenocytes do not exhibit PHA-stimulated growth in FBS-supplemented medium, they are reproducibly stimulated in this serum-containing medium by phorbol 12-myristate, 13-acetate (PMA). This suggests that FBS may interfere with (or does not support) some early step(s) in lectin-induced signalling, rather than with proliferation itself.     

Hierarchy of Stroma-derived Factors in Supporting Growth of Stroma-dependent Hemopoietic Cells: Membrane-bound SCF is Sufficient to Confer Stroma Competence to Epithelial Cells

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34 + cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34 + hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.     

Differentiation of human embryonic stem cells in serum-free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis

We have utilized a serum- and stromal cell-free "spin embryoid body (EB)" differentiation system to investigate the roles of four growth factors, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), and basic fibroblast growth factor (FGF2), singly and in combination, on the generation of hematopoietic cells from human embryonic stem cells (HESCs). Of the four factors, only BMP4 induced expression of genes that signaled the emergence of the primitive streak-like population required for the subsequent development of hematopoietic mesoderm. In addition, BMP4 initiated the expression of genes marking hematopoietic mesoderm and supported the generation of hematopoietic progenitor cells at a low frequency. However, the appearance of robust numbers of hematopoietic colony forming cells and their mature progeny required the inclusion of VEGF. Finally, the combination of BMP4, VEGF, SCF, and FGF2 further enhanced the total yield of hematopoietic cells. These data demonstrate the utility of the serum-free spin EB system in dissecting the roles of specific growth factors required for the directed differentiation of HESCs toward the hematopoietic lineage.     

Serum inhibitors for human mast cell growth: possible role of retinol

BACKGROUND: In vitro culture systems have been used to study the physiological and pathological characteristics of human mast cells. However, there are some differences in proliferation and maturation of mast cells between fetal bovine serum (FBS)-containing and serum-deprived cultures. Accordingly, we attempted to identify circulating factor(s) affecting the development of human mast cells. METHODS: We measured the serum levels of retinol and several cytokines. To elucidate the antiproliferative effects of the serum, a retinoic acid receptor (RARalpha) antagonist and neutralizing antibodies against cytokines were used. RESULTS: Similar to FBS, human serum dose-dependently suppressed the growth of tryptase+ cells from CD34+ cord blood cells or 20-week cultured mast cells under stimulation with stem cell factor (SCF). The serum-mediated inhibition might be based on a decline in proliferation rate. Among inhibitors for mast cell growth, retinol and transforming growth factor (TGF)-beta1 were present at high levels in human serum. In contrast with anti-TGF-beta1 antibody, an RARalpha antagonist counteracted the serum-induced suppression of human mast cell proliferation. CONCLUSIONS: Our results suggest that retinol and its derivatives act as a circulating regulator for human mast cell growth. The RARalpha antagonist may be a useful tool to obtain higher numbers of mast cells in FBS-containing cultures.     

HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells

Hepatocyte growth factor (HGF) is a multifunctional cytokine with early hematopoiesis-stimulatory activity. Here, we focus on its erythropoiesis-stimulatory effect on highly purified human hematopoietic progenitor cells (CD34+/CD45+ cells) derived from the cord blood. In immunoblot analyses, c-met protein (a receptor of HGF) was detected in the CD34+/CD45+ cells, although the expression levels were different among samples. The c-met expression was facilitated by incubation of the cells with stem cell factor (SCF) or interleukin 3 (IL-3), even if the expression level had been low. IL-6, G-CSF, or erythropoietin (EPO) did not show such a stimulatory effect on the c-met expression of the cells. When HGF was added to the CD34+/CD45+ cells in the presence of SCF, the numbers of CD36+/CD11b- cells (very early erythroid lineage cells) and BFU-E increased. EPO-dependent tyrosine phosphorylation of Stat 5 also increased, but the EPO receptor (EPO-R) expression remained unchanged in the CD34+/CD45+ cells treated with SCF + HGF. Our present study suggests that stimulation of the HGF/c-met signal is concomitant with induction of c-met protein by SCF. The subsequent enhancement of signal transduction via the activation of Stat 5 from the EPO-R plays a crucial role in the commitment of hematopoietic stem cells into erythroid lineage cells.     

Enhancement of hybridoma production by medium supplemented with murine ascitic fluid

We have greatly improved the yield after cell fusion of antigen-specific monoclonal antibody-secreting murine hybridomas by substitution of Sp2/0 ascites for fetal bovine serum (FBS) in the culture medium. As a medium supplement for established cultures, Sp2/0 ascites at 2.5% in Dulbecco's modified Eagle's medium (DMEM) provided growth characteristics similar to media containing 10% or 20% FBS. All culture parameters associated with hybridoma fusion experiments, however, were advantageously affected in ascites-supplemented cultures. Clonal growth of hybridomas from the single cell stage was enhanced at least two-fold over 20% FBS-supplemented medium. Following fusion, both the number of colonies and hybridoma growth rates were substantially increased for ascites-containing cultures. Most importantly, the number of antigen-specific antibody-secreting hybridomas was increased in Sp2/0 ascites supplemented cultures, five-fold in the eight fusion experiments presented here. This improved performance compared to FBS-supplemented medium is reproducible from lot to lot of ascites.     

细胞因子短期扩增对造血干/祖细胞黏附和迁移能力的影响

目的：探讨干细胞因子（SCF）+白细胞介素-6（IL-6）短期扩增对CD34+造血干/祖细胞黏附和迁移能力的影响。方法:用密度剃度离心的方法分离脐血CD34+细胞，经SCF和IL-6孵育48 h，用CCK-8方法检测CD34+细胞增殖能力；用流式细胞仪检测处理前后的CD49d（VLA-4）、CD11a（LFA-1）、CD62L（L-selectin）及CD184（CXCR4）的表达。用纤连蛋白（FN）包被96孔板，检测经或未经因子扩增的CD34+细胞的黏附能力。扩增的CD34+细胞悬浮于transwell培养板的上层，下层添加基质细胞衍生因子（SDF-1），流式细胞仪检测迁移细胞数，计算迁移率。结果:经SCF+IL-6处理48h后CD34+细胞扩增近3倍；表达CD49d、CD11a、CD62L及CD184的CD34+细胞的百分数分别由原来的26.34%±5.37%、17.63%±4.57%、46.38%±6.61%和9.58%±1.56%增加到65.67%±8.72%、56.67%±6.34%、84.76%±9.57%和19.32%±3.64%（P<0.01）。扩增后的CD34+细胞对FN的黏附能力及在SDF-1诱导下的迁移作用都显著增强（P<0.01）。结论:SCF+IL-6短期扩增CD34+ 造血干/祖细胞显著增加细胞的黏附能力，增加SDF-1诱导的迁移作用，可能是SCF+IL-6促进归巢的主要机制之一。     

Culturing of BHK-21 cells in a medium containing adult bovine serum and pituitary extract

Summary To achieve a comparable protein and cell production by culturing of BHK-21 cells in monolayer, in a medium containing adult bovine serum (ABS) and in a medium containing fetal bovine serum (FBS), 1.5 times (v/v) more ABS than FBS has to be added. At a volume fraction in the medium of less than 2% ABS, no proliferation is observed. For a fixed protein and cell production, the amount of required protein in a medium containing ABS can be reduced by addition of a protein extract from pituitary glands from calves (PEP). The relative cell density after 7 days in a culture medium containing 2 or 5% (v/v) ABS is 3 times higher when 0.2 g/liter pituitary extract is added. This indicates a synergistic interaction between components from ABS and from the protein extract. The addition of a pituitary extract to a medium with ABS also induces a prolonged cell proliferation period. A combined addition to a medium with ABS of 0.02 g/liter PEP and 0.15 g/liter heparin yields almost the same cell density as the addition of 0.2 g/liter PEP alone, indicating the presence of fibroblast growth factor (FGF) in the pituitary extract. However, the enhanced cell production by the addition of a pituitary extract to a medium containing ABS cannot be explained only by the presence in this crude extract of a- or bFGF.Abbreviations ABS adult bovine serum - BE protein extract from whole bovine brains - FBS fetal bovine serum - PEP protein extract from pituitary glands from calves - PEX purchased bovine pituitary extract - PDT population doubling time     

Comparison of a serum replacement (Omni Serum) and fetal bovine serum in cell cultures used to isolate herpes simplex virus from clinical specimens.

Traditionally, fetal bovine serum (FBS) has been the principal component in media used in the growth and maintenance of cell cultures. Recent shortages have affected the cost and availability of FBS to clinical laboratories. Furthermore, lot-to-lot variability can affect cell culture performance and growth. We evaluated a commercially available serum replacement (Omni Serum; Advanced Biotechnologies Inc., Columbia, Md.) for use in the growth of cell cultures and for use in maintenance media used for the isolation of herpes simplex virus from clinical specimens. Cells (rhabdomyosarcoma and mink lung) raised on 5% Omni Serum grew as well as those grown on 10% FBS. The sensitivity of the Omni-raised cells to herpes simplex virus that had been isolated from 111 clinical specimens was equal to that of the cells raised and maintained with FBS. Cells grown with 10% FBS and maintained with 2% Omni Serum displayed the same sensitivity and integrity in tubes (rhabdomyosarcoma and mink lung) and vials (MRC-5 cells) as cells grown with 10% FBS and maintained with 5% FBS. This study indicates that Omni Serum is an acceptable substitute for FBS in maintenance media for cell culture tubes and vials used for viral isolation from clinical specimens.     

合成多肽为半抗原制备抗SCF受体单克隆抗体

本文成功地建立了以合成的小分子肽与蛋白质交联的偶联物为免疫原，制备单克隆抗体（ｍＡｂ）的技术路线。选定ＳＣＦ受体特异性肽段进行人工合成，将其偶联于ＢＳＡ免疫动物，应用细胞融合技术制备了抗两肽段的杂交瘤细胞７株。ＥＬＩＳＡ法和免疫印迹结果显示，其分泌的ｍＡｂ特异性强，与偶联载体及多种多肽生长因子均无交叉反应。经ＡＰＡＡＰ法鉴定，抗胞内羧基端肽的ｍＡｂＳＢ３０４与ＳＣＦ受体表达细胞系ＨＥＬ有弱的阳性反应；免疫组化测定与胎儿皮肤基底层色素细胞及胃粘膜上皮细胞呈阳性反应，提示ｍＡｂＳＢ３０４可识别天然状态的ＳＣＦ受体。     

Differences in the effect on neural stem cells of fetal bovine serum in substrate-coated and soluble form

The influence of fetal bovine serum (FBS) adsorbed to poly(ethylene-co-vinyl alcohol) (EVAL) and polyvinyl alcohol (PVA) substrates (coated FBS) and FBS present in the culture medium (soluble FBS) on the behavior of embryonic rat cerebral cortical neural stem cells was studied at neurosphere level. When both coated FBS and soluble FBS were not present in the culture system, the fate and behavior of neurospheres were mediated mainly by the substrates used. When neurospheres were cultured either on FBS-coated EVAL or FBS-coated PVA substrates in the serum-free medium, the most striking morphological characteristic of neurospheres was that these neurosphere-forming cells attached and were induced to differentiate into process-bearing cell phenotypes predominantly; however, the differentiated cell phenotypes were dissimilar on these two substrates. On the contrary, when neurospheres were cultured in the medium containing 10% FBS, the neurosphere-forming cells were induced into protoplasmic cells typically but no difference in differentiated cell phenotypes on EVAL and PVA substrates was observed. Interestingly, instead of promoting process outgrowth under serum-free medium condition, coated FBS enhanced migration of differentiated protoplasmic cells when soluble FBS were present. These results inform that the substrates, coated serum, and soluble serum within the culture environment together can significantly alter cell behavior and morphological differentiation and will therefore be an important clue for the development of biomaterials to regulate the potential of the CNS neural stem cells.     

Induction of the Expression of SCF in Mouse by Lethal Irradiation

Abstract

To clarify what kinds of cytokines are actually contributing to proliferation of hemopoietic stem cells in vivo after lethal irradiation, we have investigated the expression of some cytokines by RT-PCR method. Above all, expression of the SCF was increased significantly in the bone marrow cells soon after lethal irradiation in both the Sca-1 (+) bone marrow cells injected and non-injected mice. The day 6 serum from the lethally irradiated mice could support the proliferation of the Sca-1 (+) bone marrow cells, even though the serum from normal mice could not. The quantification analyses have revealed the increase of the amounts of IL-6 and flt3-ligand in their serum, but not significant increase of the amount of SCF. Precise PCR analysis has revealed that the cell surface associated form of SCF was significantly induced in the bone marrow after lethal irradiation. These data indicate that the cell surface form of SCF mainly promotes the proliferation of hemopoietic stem cells with some soluble cytokines under sever lack of hemopoietic stem cells in vivo caused by lethal irradiation and also suggest the importance of direct cell-to-cell interaction on proliferation of hematopoietic stem cells in vivo.     

Monoclonal antibodies to nerve growth factor from bovine seminal plasma

Mouse monoclonal antibodies directed against nerve growth factor (beta NGF) from bovine seminal plasma have been isolated and characterized. They are produced by hybridomas derived from Sp2/0.Ag14 myeloma cells and spleen cells from BALB/c mice immunized with beta NGF which was purified by the method of Harper et al. [J. biol. Chem. 257, 8541-8548 (1982)]. Five of these hybridomas can be grown in ascites tumor form and secrete antibodies of the IgG1 or IgG2a subclass. When used to probe the components of seminal plasma extracts or purified beta NGF as separated electrophoretically on SDS gels, the antibodies react with the beta NGF band at Mr = 15,000. The antibodies bind to native bovine beta NGF, but bind very poorly to mouse beta NGF. Antibody exclusion and additive-binding experiments indicate that these antibodies bind to the 1 antigenic domain. The cell receptor binding site is probably not close to this domain, as the antibodies fail to block the biological activity of bovine beta NGF on cultures of dissociated neurons from sensory ganglia. These monoclonal antibodies define a region in which bovine beta NGF is structurally different from the closely related molecule mouse beta NGF.     

Fetal bovine serum contains an inhibitor of interleukin-1

The keratinocyte cell line COLO-16 constitutively produces factors with interleukin-1 (IL-1) activity including IL-1 alpha and IL-1 beta. IL-1 activity assayed by thymocyte proliferation from cell supernatants was 20-50% less if cells were maintained in media containing 10% fetal bovine serum (FBS) compared to media without serum 24 h prior to harvest. The increased IL-1 activity in supernatants from cells in serum free media was not due to increased cellular levels of IL-1 alpha or IL-1 beta mRNA. Similarly, IL-1 activity recovered from conditioned supernatants of COS cells transfected with expression vectors containing IL-1 beta cDNA was approximately 22-45% less in cells grown in 20% FBS medium compared to similar cultures grown for 3 days post transfection in 1% FBS. When serial dilutions of recombinant IL-1 were made in buffer containing 10% FBS and assayed by a thymocyte proliferation method, a 30-50% decrease in activity was observed. IL-1 activity was also measured by its ability to induce prostaglandin E2 synthesis by fibroblasts. When COS conditioned supernatants were applied to fibroblast cultures there was 30% less prostaglandin E2 activity from fibroblasts treated with COS supernatants containing 20% FBS, compared to supernatants containing 1% FBS. The inhibitor molecule was partially purified by gel filtration and found to have a molecular weight of approximately 85,000. The presence of FBS in cell-conditioned media significantly reduces the sensitivity of IL-1 detection by bioassay techniques.