Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue

Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.     

Proliferation of hemopoietic stem cells in culture of embryonal liver of mice

The vinblastine technique was used to demonstrate that the proliferative capacity of colony-forming cells in organ culture of embryonal liver of mice between the first and seventeenth day of cultivation is adequately high. The relative content of CFU increases in culture with a maximum between one and two weeks.     

Isolation of hemopoietic pluripotent stem cells from spleens of thiamphenicol-pretreated mice

The present report describes the bulk isolation of pluripotent stem cells (PSC) (assayed as day-9 CFU-S, colony-forming-units-spleen). As starting material, spleens, highly enriched with PSC, were used from mice that were bled and treated with thiamphenicol (TAP). In subjecting the spleen cells to a two-stage centrifugal elutriation procedure and a subsequent Percoll gradient centrifugation stage a 30-fold enrichment in the CFU-S concentration was achieved. The splenic PSC seeded with a characteristic low efficiency in the spleens of irradiated mice (f = 2%). Correcting the colony number for this, we obtained a cell mixture consisting of 88% PSC, contaminated with 4% committed precursor cells and about 10% ganuloid cells.     

Growth kinetics of hemopoietic fetal stem cells

Fetal spleen stem cells have growth characteristics similar to those of normal adult spleen stem cells. On the contrary there is an early fetal liver stem cell population which possesses a lag time longer than that of adult stem cells. The duration of the lag time is controlled by a built-in biological timer which seems to regulate some proliferative functions of the primitive liver stem cell.     

Pluripotent hemopoietic stem cells in mice and humans

Although it has been reported previously that pluripotent hemopoietic stem cells (P-HSCs) express c-kit, the receptor for stem cell factor (steel factor), we and other groups have recently shown that P-HSCs do not express c-kit. In this review, we provide evidence that c-kit 2 years) and the capacity to form colony-forming units in spleen (CFU-S) on Day 16, although c-kit(low) HSCs or c-kit+ HSCs have LTRA less than 1.5 years and the capacity to form CFU-S on Day 14 or on Day 10, respectively. In addition, we have found that there is a major histocompatibility complex (MHC) restriction between P-HSCs and stromal cells; normal P-HSCs can proliferate and differentiate efficiently in collaboration with MHC class I-compatible (but not MHC class I-incompatible) stromal cells. In humans, we also show that c-kit相似文献   

Temporal and spatial localization of osteoclasts in colonies from embryonic stem cells

Osteoclasts are hematopoietic cells essential for bone resorption. To understand the process of osteoclastogenesis, we have developed a culture system that employs a stromal cell line, in which differentiation of osteoclasts from single embryonic stem (ES) cells occurs. This culture, which did not require any cell passaging or other manipulations, enabled us to investigate the temporal and spatial localization of the osteoclast lineage in the colonies formed from ES cells. Cells expressing tartrate-resistant acid phosphatase, a specific marker of the osteoclast lineage, were first detected on day 8, and subsequently became localized at the periphery of colonies and matured into multinucleated cells to resorb bone. Addition of macrophage colony-stimulating factor and osteoprotegerin-ligand, which are produced by stromal cells, promoted osteoclastogenesis in whole colonies, indicating that the location and maintenance of mature osteoclasts as well as the growth and differentiation of osteoclast precursors are regulated by these two factors.     

A stochastic model of self-renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture

We recently identified a murine hemopoietic stem cell colony which consists of undifferentiated (blast) cells and appears to be more primitive than CFU-GEMM in the stem cell hierarchy. The progenitors for the colony which we termed “stem cell colony” possess an extensive self-renewal capacity and the ability to generate many secondary multipotential hemopoietic colonies in culture. We replated a total of 68 stem cell colonies from cultures of murine spleen cells and analyzed the number of stem cell–and granulocyte(neutrophil)-erythrocyte-macrophage-megakaryocyte (GEMM) colony-forming cells in individual stem cell colonies. Of the 68 stem cell colonies, 35 contained progenitors (abbreviated as “S”-cells) for stem cell colonies. The distributions of S-cells and CFU-GEMM in individual stem cell colonies were extremely heterogeneous. Neither the frequency distributions of S-cells nor CFU-GEMM in stem cell colonies could be fitted well by Poisson distribution. Rather, the frequency distribution of the s-cells could be approximated by a geometric distribution and that of CFU-GEMM by an exponential distribution, both of which are variates of the gamma distribution. Our observations are in agreement with those on the distributions of CFU-S in individual spleen colonies and provided support for a stochastic model for stem cell self-renewal and commitment in culture. Application of the theory of the branching process to the distribution of S-cells revealed a distributional parameter “p” of 0.589 which is also in agreement with the earlier report on the p value for reproduction of CFU-S.     

The self renewal probability of hemopoietic stem cells

The probability of a colony which originated as a single stem cell to become extinct due to differentiation of all of its stem cells in any generation is closely connected to stem cell self renewal probability p. p can be determined from the coefficient of variation of the colony numbers received by reinjecting single colonies of the same age. Whole spleens containing a known average colony number can also be used with advantage for this purpose. The results of both procedures indicate a stem cell self renewal probability p =0.62 ± 0.04, which does not change significantly between the sixth and the fourteenth day of colony development, and an extinction probability ω = 0.63 ± 0.12.     

Serum-free culture of primitive murine hemopoietic colony-forming cells

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.     

The cellular composition of hemopoietic spleen colonies

The cellular composition of individual hemopoietic spleen colonies has been studied using techniques which tested primarily for cell function rather than cell morphology. Erythroblastic cells were recognized by their capacity to incorporate radioiron, granulocytic cells by their content of peroxidase-positive material, and hemopoietic stem cells by their ability to form spleen colonies in irradiated hosts. It was found that, 14 days after the initiation of spleen colonies, the distribution of these cell types among individual colonies was very heterogeneous, but that most colonies contained detectable numbers of erythroblasts, granulocytes and colony-forming cells. An appreciable proportion of the cells in the colonies could not be identified as any of these three cell types. No strong correlations between numbers of erythroblasts, granulocytes and colony-forming cells in individual colonies were observed, though there was a tendency for colonies containing a high proportion of erythroblasts to contain a low proportion of granulocytes, and for colonies containing a high proportion of granulocytes to contain a higher proportion of colony-forming cells. An analysis of colonies which contained cells bearing radiation-induced chromosomal markers indicated that 83–98% of the dividing cells within 14-day spleen colonies were derived from single precursors.     

Identification and characterization of splenic adherent cells forming densely-packed colonies

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.     

The in vitro radiosensitivity of hemopoietic stem cells from control and preirradiated infant mice

Summary The radiosensitivity of hemopoietic stem cells isolated from infant mice (6 or 9 days of life), of infant preirradiated mice (exposed to 126 rad on day 6 and assayed at day 9 of life) and of adult C57/B1 mice was assayed on the basis of their capacity to form spleen colonies and to incorporate iododeoxyuridine after transplantation into heavily irradiated hosts. Stem cells of infant non-irradiated mice have a D₀ of 115 rad compared to 72 rad for adult mice whereas the D₀ of preirradiated infant mice has diminished to 80 rad. No significant difference in D₀ was seen between spleen and bone marrow cells or between total cells and cells not sensitive to³H-thymidine. It is postulated that this sensitization of stem cells caused by a preirradiation is responsible for the greater mortality of infant mice after fractionated exposure compared to a single one.