Growth of a pure population of mouse mast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes.

A pure population of mast cells was obtained after 14 days of culturing mouse bone marrow cells in the presence of medium derived from concanavalin A-stimulated mouse spleen cells. The cells were characterized as mast cells by their morphologic appearance and histologic staining, by their histamine content (450 ng per 10(6) cells) and by the demonstration of IgE receptors on their surface (150,000--440,000 receptor sites per cell). The histamine content and the number of IgE receptors remained constant for at least 7 wk of culture. These mast cells could be passively sensitized to mice hybridoma IgE. They then released 43% of their histamine content upon incubation with anti-mouse hybridoma IgE.     

Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin.

Cells from CBA fetal mouse liver formed pure or mixed erythroid colonies in semisolid agarculture after stimulation by medium conditioned by pokeweed mitogen-stimulated mouse spleen cells. In general shape, the erythroid colonies resembled typical 7-day single or multiple (burst) colonies. However one-third to one-half contained, in addition to erythroid cells, macrophages and neutrophils and, less commonly, megakaryocytes or eosinophils. Culture of micro manipulated single colony-forming cells showed these erythroid colonies to be clones. Colony-forming cells declined in frequency with advancing fetal age, but low numbers were detectable in adult bone marrow. Assays of spleen conditioned medium in polycythemic mice failed to detect erythropoietin; the cloning system may detect a fetal type of erythropoietin-independent, erythropoietic cell since few were detected in adult marrow.     

THE ROLE OF INDIVIDUAL SPLEEN CELLS IN THE INTERFERON RESPONSE OF THE INTACT MOUSE

A method is described for the detection of interferon production by individual spleen cells of mice after intravenous virus infection. Although mouse spleen plays a major role in the total interferon response to Newcastle disease virus, the number of spleen cells participating is a small fraction of the total, suggesting that interferon formation is to some extent a specialized cell function.Monolayers of mouse embryo cells, after brief contact with agar-suspended spleen cells from interferon-producing mice, have roughly circular foci of cells remaining after removal of agar and vesicular stomatitis virus challenge. The numbers of such foci correlate directly with the number of spleen cells, concentration of inducing virus in the inoculum, duration of contact of cells with mouse-embryo indicator monolayers, time after interferon induction, and serum interferon titer. With this technique, evolution of the interferon-forming cell response in spleens of virus-infected mice has been studied.     

Properties of Adenylate Cyclase of Lymphoid Cells

Adenylate cyclase of homogenates or lysates of mouse and rat lymphoid tissues was activated by the addition of fluoride ion, epinephrine, or norepinephrine, but not by any of several other hormones. The catecholamine stimulation was characterized as beta-adrenergic. This activity was localized in the small lymphoid cells, was greater in thymic than in splenic or mesenteric node cells, and also was greater in mouse than in rat cells. Catecholamine-stimulated activity of mouse thymocytes remained constant from the 17-19th day of fetal development to 5 weeks after birth; it subsequently decreased to about one-half of the activity by 7-8 weeks. Similar decreases with age occurred in mouse spleen and rat thymus. In contrast, the glucagon-stimulated activity of rat liver increased during a similar period. Hypophysectomy of rats at 3 weeks did not influence the amount of cyclase activity of thymocytes assayed at 7 weeks.When intact mouse thymocytes were first incubated in a culture medium at 37 degrees C with epinephrine or norepinephrine, a second addition of catecholamine after cell lysis no longer stimulated the enzymes. This loss of stimulation was selective, since basal activity and stimulation by fluoride ion were not affected. Incubation of intact cells with phytohemagglutinin markedly decreased the activity of lysates, whether assayed in the presence or absence of catecholamine or fluoride. In contrast, phytohemagglutinin added directly to the assay had no effect. No alternations occurred in adenylate cyclase activity as a result of the incubation of a 1:1 mixture of thymocytes from two strains of mice selected for the capacity of the cells to produce a mixed lymphocyte response.     

Hemoglobin switching during murine embryonic development: evidence for two populations of embryonic erythropoietic progenitor cells

Explants of normal mouse embryonic tissues and disaggregated embryonic single cells were cultured in vitro to study the erythropoietic progenitor cells present during embryonic development. The results indicate that there are two populations of erythropoietic progenitor cells committed to different hemoglobin synthetic programs. These progenitor cells are present at an early gestational stage prior to the formation of the fetal hepatic primordium. One population of progenitors can be stimulated by erythropoietin alone to form usually small erythroid colonies after culture for six days in vitro. These erythroblasts primarily synthesize embryonic hemoglobins, but produce some adult hemoglobins as well. The other population of progenitors requires stimulation by both erythropoietin and adult spleen cell- conditioned medium, and usually forms large erythroid colonies after culture for six days in vitro. These erythroblasts produce only adult hemoglobins.     

Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4–19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4–6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.     

A Factor That Can be Used to Regulate an In Vitro Primary Immune Response

A factor has been isolated that supports the stimulation of an immune response against heterologous erythrocyte antigens in mouse spleen cells cultured in a medium that contains deficient serum. The factor is secreted by certain spleen cells that are obtained in permanent culture after leukemia virus infection. The factor permits antigen-sensitive cells derived from bone marrow to mature to antibody-forming cells. This factor can be used in controlling the initiation of a primary immune response in vitro.     

日本血吸虫虫源性抗原诱导效应性B细胞分化的研究

目的 目的 观察日本血吸虫可溶性虫卵抗原 （SEA） 和可溶性成虫抗原 （SWAP） 诱导小鼠效应性B细胞分化情况。方 方 法 法 SEA、 SWAP和脂多糖 （LPS） 分别体外刺激小鼠脾脏单个核细胞和脾脏CD19+ B细胞, 72 h后采用流式细胞术检测 CD19+ IL?6+ 及CD19+ IFN?γ+ 细胞比例; 用酶联免疫吸附试验 （ELISA） 检测抗原刺激后脾脏B细胞培养上清中IL?6和IFN?γ 水平。用SEA、 SWAP、 PBS分别和不完全弗氏佐剂混合免疫小鼠, 每周1次, 共3次, 末次免疫后7 d取小鼠脾脏, 流式细胞 术检测CD19+ IL?6+ 及CD19+ IFN?γ+ 细胞比例, 用ELISA法检测培养上清中IL?6和IFN?γ水平。 结果 结果 SEA与LPS可以体 外诱导脾脏单个核细胞和脾脏B细胞分化为CD19+ IL?6+ B细胞 （F = 5.099, P < 0.05; F = 6.951, P < 0.05）, 并刺激脾脏B细 胞分泌IL?6 （F = 55.184, P < 0.01）; SEA免疫小鼠后, 可以在体内刺激小鼠脾脏B细胞分泌IL?6 （F = 19.244, P < 0.01）, 并诱 导小鼠脾脏单个核细胞分化为CD19+ IL?6+ B细胞 （F = 14.904, P < 0.05）。SWAP可以体外诱导脾脏单个核细胞分化为 CD19+ IFN?γ+ B细胞 （F = 3.277, P < 0.05）, 但不能单独诱导脾脏B细胞分化为IFN?γ+ B细胞, 也不能刺激脾脏B细胞分泌 IFN?γ; SWAP免疫小鼠后, 可以在体内刺激小鼠脾脏B细胞分泌IFN?γ （F = 31.886, P < 0.01）, 并诱导其分化为CD19+ IFN? γ+ B细胞 （F = 49.873, P < 0.01）。 结论 结论 日本血吸虫SEA可以优势诱导小鼠脾脏单个核细胞分化为CD19+ IL?6+ B细胞, 而 SWAP可以优势诱导小鼠脾脏单个核细胞分化为CD19+ IFN?γ+ B细胞, 但依赖于其他免疫细胞的参与。     

Abnormalities in myelopoietic regulatory interactions with acidic isoferritins and lactoferrin in mice infected with Friend virus complex: association with altered expression of Ia antigens on effector and responding cells

The regulation of myelopoiesis was evaluated in B6D2F1 mice inoculated with Friend virus complex (spleen focus-forming virus plus helper virus) or helper virus alone by analyzing acidic isoferritin (AIF) and lactoferrin (LF) interactions with target cells. Under normal conditions, AIF suppresses colony and cluster formation by an Ia- antigen-positive cycling subpopulation of mouse granulocyte-macrophage progenitor cells (CFU-GM). Under the same conditions, the release of AIF-inhibitory activity and granulocyte-macrophage colony stimulatory factors (GM-CSF) from an Ia-antigen-positive subpopulation of monocytes and macrophages is suppressed by LF. Within one to two days after inoculation in vivo with Friend virus complex or helper virus, mouse CFU-GM become insensitive in vitro to suppression by purified human AIF as well as crude mouse AIF, and by four days, bone marrow, spleen, and thymus cells of these mice release much greater quantities of AIF- inhibitory activity than the cells from mice injected with control medium. The Friend virus complex itself has no influence in vitro on CFU-GM from normal mice. In addition, the release of AIF-inhibitory activity from bone marrow, spleen, and resident peritoneal cells and the release of GM-CSF from resident peritoneal cells of mice infected with Friend virus complex are not suppressed by LF. The inability of AIF to suppress colony formation by bone marrow and spleen CFU-GM from mice infected with Friend virus complex is associated with the loss of Ia (I-A subregion) antigens from CFU-GM, even though CFU-GM are in cycle. The nonresponsiveness of bone marrow, spleen, and peritoneal cells from these mice to LF suppression of AIF release and the inability of LF to influence GM-CSF release from peritoneal cells is associated with loss of Ia antigens from these cells. The above abnormalities are similar to the defects noted using cells from patients with leukemia. These results suggest that mice infected with Friend virus complex can serve as a model for investigating abnormalities in cell regulation and their relationships to disease progression.     

Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three- dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3-2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units- erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3-2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3-2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.     

Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines.

Multipotential hematopoietic progenitor cell lines have been established from nonadherent cell populations removed from continuous mouse bone marrow cultures. Clonal sublines of lines B6SUtA or B6JUt derived from single cells formed mixed colonies containing erythroid cells, neutrophil-granulocytes, and basophil/mast cells in semisolid medium containing erythropoietin and conditioned medium from pokeweed mitogen-stimulated spleen cells. Each of several subclones of cell line Ro cl formed colonies containing eosinophils, neutrophil-granulocytes, and basophil/mast cells in semisolid medium. Multipotentiality was maintained in vitro for over 2 1/2 years. In contrast, cell line 32D formed basophil/mast cell colonies with no detectable differentiation to other pathways. Multipotential cell lines did not produce detectable spleen colonies (CFUs) in vivo, nor did intravenous inoculation of up to 5 X 10(7) cells protect lethally irradiated mice from bone marrow failure. Newborn and adult mice inoculated with 5 X 10(7) cells showed no detectable leukemia or solid tumors after one year. Both multipotential and committed basophil/mast cell lines demonstrated absolute dependence upon a source of a growth factor(s) found in medium conditioned by WEHI-3 cells. These cell lines should be of value in studies of the regulation of hematopoietic stem cell differentiation in vitro.     

Enhancement of the primary cytotoxic response to membrane by a lymphokine costimulator.

The primary and secondary cytotoxic T cell responses of C57Bl/6 (H-2b) mouse spleen cells to P-815 membrane fragments (H-2d) were examined. The primary response required the addition of a supernatant from mitogen-stimulated spleen cells, or costimulator, to the culture medium. Costimulator had little effect on the secondary response unless the spleen cells were first passed through a nylon wool column. Our data suggested that secondary cultures produced their own costimulator and that adherent cells were required for its production. The possibility that adherent cells are needed to activate helper cells is discussed.     

Host immune responses after administration of inactivated Venezuelan equine encephalomyelitis virus vaccines. II. Kinetics of neutralizing antibody responses in donors and adoptively immunized recipients.

Lymphoid cell responses to immunization with various formalin-inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccines were monitored in mice by assessment of the development of both the neutralizing antibody response in sera of spleen cell donors and the adoptive neutralizing antibody response induced by spleen cell transfer in recipients. Donors immunized intraperitoneally with formalin-inactivated VEE vaccine (a single dose or a dose on three consecutive days) developed early serum neutralizing antibody responses (larger than or equal to 1:88-1:100) by seven days after immunization. Recipients of spleen cells from such mice were, however, incapable of eliciting a neutralizing antibody response (less than or equal to 1:10). Only spleen cells from donors immunized with inactivated VEE vaccine plus adjuvants (particularly complete Freund's adjuvant and Bordetella pertussis) were consistently capable of producing early, high-titer serum neutralizing antibody responses in adoptively immunized recipients (larger than or equal to 1:50-1:120 on day 4). The magnitude of neutralizing antibody responses of donors to inactivated VEE vaccines did not serve as a useful indicator of whether spleen cells from such mice could adoptively induce antibody responses in recipients. Finally, treatment of immune spleen cells with rabbit antiserum to mouse thymocytes, but not with rabbit antiserum to mouse gamma-globulin or normal rabbit serum, abolished the capacity of such cells to transfer an antibody response adoptively.     

Modification of antisense phosphodiester oligodeoxynucleotides by a 5'' cholesteryl moiety increases cellular association and improves efficacy.

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.     

Adult hemoglobins are synthesized in erythroid colonies in vitro derived from murine circulating hemopoietic progenitor cells during embryonic development.

Circulating peripheral blood cells and disaggregated yolk sac cells were obtained from normal mouse embryos as early as day 9 of gestation, prior to the formation of the fetal liver. These were cultured in vitro in plasma clots or methylcellulose, in the presence of either embryonic fluid or adult spleen cell-conditioned medium, with or without added erythropoietin. Large erythroid colonies were observed by the sixth day of culture. In all instances, these erythroid colonies synthesized adult hemoglobins. These results indicate that erythroid progenitor cells committed to adult hemoglobin synthesis are present in early embryonic circulation.     

Binding of autologous erythrocytes to immature T-cells.

A small percentage of normal mouse thymus and spleen lymphocytes form rosettes with autologous erythrocytes. The number of these autologous rosettes increases 15- to 20-fold after adult thymectomy and to a lesser degree with aging. Autologous rosette level is also abnormally high in nude (congenitally athymic) mice. The high level of autologous rosette-forming cells found after adult thymectomy is normalized by injecting ng amounts of purified circulating thymic factor. Autologous rosette-forming cells adhere to nylon, belong to the less dense spleen cells, are in majority steroid-resistant in the thymus. All these properties suggest that autologous rosette-forming cells might belong to immature T-cell (thymic-dependent cell) precursors.     

The Growth of Bone Marrow Cells in Liquid Culture

S ummary . Liquid suspension cultures of mouse bone marrow cells at high and low density were prepared in supplemented Eagle's medium containing 10% of a partially purified extract of mouse embryos and pregnant mouse uterus (PMU). In the low cell density cultures the number of cells decreased for 2 days; by 4 days the agar colony-forming cells (agar CFC) had risen ten-fold and the spleen colony-forming units (spleen CFU) had fallen to one tenth; between 4 and 8 days the total cell count showed a four-fold increase and the final cell number exceeded the number of the original culture. The cells produced were mainly macrophages. If PMU was not included in the culture the agar CFC disappeared after 4 days and there was no cell multiplication. In the high cell density cultures a similar pattern was observed; in the presence of PMU the agar CFC showed an increase in number, the spleen CFU decreased and an increase in cell number occurred between 4 and 8 days. However, the cells produced were predominantly granulocytes. In the absence of PMU from this cell culture, agar CFC were maintained for 6 days and the cell population remained predominantly granulocytic. These methods of growing cell enable cell recovery from the cultures to be made at any stage and provide an opportunity to study the kinetics and functional capacity of the cells produced.     

伯氏疟原虫氯喹抗性株感染ICR小鼠脾脏树突状细胞成熟和B细胞活化

目的 研究伯氏疟原虫氯喹抗性株(RC株)和氯喹敏感株(N株)感染鼠脾脏B细胞活化与树突状细胞(DC)的关系。 方法 分别用感染N株或RC株疟原虫的红细胞(iRBC)腹腔接种感染ICR小鼠(1×10⁶个iRBC/只)。当小鼠原虫血症N株达50%～80%、 RC株达61.7%～68.4%时, 断颈处死取脾脏。 常规方法制作石蜡切片, HE染色或免疫组织化学染色, 进行组织学观察。 制作超薄切片, 透射电镜观察脾脏细胞的变化。制作冰冻切片进行免疫荧光观察。流式细胞仪分析比较B细胞和DC变化。 结果 RC株感染小鼠脾脏白髓增生明显, 抗B细胞的特异性表面分子CD45R/B220和CD19抗体同时表达阳性的B细胞在脾细胞中的百分比增加, 中、 小淋巴细胞数量增多, 在红髓内浆母细胞与成熟的浆细胞数量增多。而N株感染小鼠脾小体则以大、 小淋巴细胞为主, 生发中心不明显, 红髓可见大量的含疟原虫的红细胞、 小淋巴细胞, 而浆母细胞和其他发育期浆细胞则少见。 RC株感染小鼠脾脏内白细胞分化抗原11c(CD11c) 阳性的DC数量明显增多, 尤其动脉周围淋巴鞘T细胞区。并且这些DC表面主要组织相容性复合体Ⅱ (MHCⅡ) 类分子表达明显升高, 表明主要是成熟的DC增多。DC外形不规则, 胞质丰富, 电子密度高, 含发达的高尔基复合体和吞噬泡样结构。 结论 RC株感染小鼠脾脏成熟的DC 明显增加, 从而诱导B细胞的活化增殖。     

Combined gene therapy of endostatin and interleukin 12 with polyvinylpyrrolidone induces a potent antitumor effect on hepatoma

AIM: To study the antitumor effect of combined gene therapy of endostatin and interleukin 12 (IL-12) with polyvinylpyrrolidone (PVP) on mouse transplanted hepatoma. METHODS: Mouse endostatin eukaryotic plasmid (pSecES) with a mouse Igkappa signal sequence inside and mouse IL-12 eukaryotic plasmid (pmIL-12) were transfected into BHK-21 cells respectively. Endostatin and IL-12 were assayed by ELISA from the supernant and used to culture endothelial cells and spleen lymphocytes individually. Proliferation of the latter was evaluated by MTT. H22 cells were inoculated into the leg muscle of mouse, which was injected intratumorally with pSecES/PVP, pmIL-12/PVP or pSecES+pmIL-12/PVP repeatedly. Tumor weight, serum endostatin and serum IL-12 were assayed. Tumor infiltrating lymphocytes, tumor microvessel density and apoptosis of tumor cells were also displayed by HE staining, CD31 staining and TUNEL. RESULTS: Endostatin and IL-12 were secreted after transfection, which could inhibit the proliferation of endothelial cells or promote the proliferation of spleen lymphocytes. Tumor growth was highly inhibited by 91.8% after injection of pSecES+pmIL-12/PVP accompanied by higher serum endostatin and IL-12, more infiltrating lymphocytes, fewer tumor vessels and more apoptosis cells compared with injection of pSecES/PVP, pmIL-12/PVP or vector/PVP. CONCLUSION: Mouse endostatin gene and IL-12 gene can be expressed after intratumoral injection with PVP. Angiogenesis of hepatoma can be inhibited synergisticly, lymphocytes can be activated to infiltrate, and tumor cells are induced to apoptosis. Hepatoma can be highly inhibited or eradiated.     

Identification in culture of a class of hemopoietic colony-forming units with extensive capability to self-renew and generate multipotential hemopoietic colonies.

Mouse marrow and spleen cells formed colonies consisting of 40-1,000 blast cells after 16 days of incubation in methylcellulose culture in the presence of medium conditioned by pokeweed mitogen-stimulated mouse spleen cells. These colonies could be distinguished from other hemopoietic colonies in situ by the complete absence of signs of terminal differentiation. Replating of these colonies (tentatively named stem cell colonies) revealed their self-renewal capacity and the extensive ability to generate secondary colonies, many of which were multipotential hemopoietic colonies. Some of the colonies revealed 100% replating efficiencies. Analyses of individual stem cells colonies revealed concurrent and high incidences of spleen colony-forming units and the macroscopic granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming units (CFU-GEMM) in culture. Replating comparison between the stem cell colonies and GEMM colonies strongly indicated that the progenitors for the stem cell colonies are higher in the hierarchy of stem cell differentiation than are CFU-GEMM. Quantitation of stem cell colonies provides an assay for the class of primitive hemopoietic progenitors described here.