Primitive Hemopoietic Stem Cells Research Articles

Blood stem cell transplantation and gene therapy of cancer.

Based on the concept of circulating hematopoietic stem cells with indefinite self-renewal capacity that gives rise to all three cell lineages, peripheral blood progenitor cells (PBPCs) have widely replaced the use of bone marrow (BM) progenitors for autologous transplantation purposes in patients with malignant hematological disorders and selected solid tumors. Ex vivo purification of normal CD34+ cell subsets contained in the patient's apheresis product possibly eliminates clonogenic tumor cells, but also serves as a target cell population for gene transduction. Genetic tagging of PBPC autografts has proven that: 1) NEOR gene expression is sustained for more than 18 months and 2) clonogenic tumor cells contaminating the autograft contribute to relapse. A second generation of gene transduction studies includes new treatment strategies such as the induction of chemoprotection (multidrug resistance gene-1), chemotherapy sensitization (p53), cancer vaccination and genetic chemosensitization. Most recently allogeneic PBPC transplantation has successfully been introduced with the intention of improving the graft-versus-leukemia effect without inducing a higher incidence or more severe graft-versus-host disease (GVHD) than what is expected after BM transplantation. Introducing the herpes virus thymidine kinase cDNA into activated donor T cells makes them susceptible to gangciclovir, thus allowing the in vivo inactivation of GVHD-inducing T cells. With the close interaction of molecular genetics and clinical oncology/hematology, genetic engineering of stem cell grafts will lead into a new stage of stem cell transplantation technology.

Interleukin 2 interacts with myeloid growth factors in serum-free long-term bone marrow culture.

IL2 infusion may benefit patients with haematological malignancies by lowering the disease burden. However, conflicting data have been reported on IL2 effects on myelopoiesis, in vitro as well as in vivo. In the present study we investigated the ability of IL2 to act on committed and primitive bone marrow progenitor cells in defined serum-free (SF) culture conditions which avoid many technical biases such as interference by exogenous stimulating or inhibiting factors. Low doses of IL2 (0.1-1000 U/ml) were studied without or in combination with recombinant IL3, GM-CSF and erythropoietin, in SF long-term marrow culture (LTMC). We report data in favour of an inhibitory activity of IL2 limited to committed progenitors and excluding more primitive haemopoietic stem cells, as shown by an alteration of CFU-GM proliferation during the first 5 weeks of LTMC, decreasing with time, unaffected BFU-E and increased nucleated cell production. Beyond week 5, no difference was observed between IL2 supplemented cultures and the SF control cultures. In parallel, IL2 induced the adherence of fibroblastic cells and their progeny. In addition to the inhibitory effect, IL2 appeared to limit the stimulating effect on granulopoiesis and erythropoiesis of myeloid growth factors (GF) such as combination of IL3, GM-CSF and EPO. Indeed, in SF-LTMC conditions, IL2 inhibitory effect is effective on CFU-GM production throughout the 7 weeks of LTMC and on BFU-E during the first 2 weeks only. These data confirm the interaction of IL2 with other GFs in the complex interplay of the cytokine network.

Self-renewal of the long-term repopulating stem cell.

Self-renewal implies maintenance of all attributes of the original in the offspring and is considered characteristic of the hemopoietic stem cells. Yet, it has been questioned whether one of the most primitive hemopoietic stem cells, the long-term repopulating cell (LTRC), has that capacity. The present experiments demonstrate that single LTRCs can repopulate the lymphohemopoietic system of a lethally irradiated mouse and that the progeny of a single LTRC in a primary recipient again contains LTRCs capable of repopulating lethally irradiated secondary recipients. The transfusion of very small numbers of marrow cells (10,000-20,000 cells containing one or no LTRCs) unexpectedly provided insight into competitive marrow repopulation. At these low levels of stem cells, irradiated host stem cells or their progeny competed successfully with unirradiated donor cells. This parallels the known reemergence and marrow repopulation by host cells when the number of nonirradiated donor stem cells is reduced by serial transplantation.

Hemopoietic stem-cell compartment of the SCID mouse: double-exponential survival curve after gamma irradiation.

It has been reported that SCID (severe combined immunodeficiency, scid/scid) mice are more radiosensitive than normal mice. In the present studies, graded doses of radiation were given to bone marrow cells from SCID mice, and double-exponential survival curves were observed for day-9 and day-12 colony-forming units in the spleen (CFU-S). Single-exponential curves were found for SCID CFU in in vitro assays for granulocyte/macrophage-CFUs and erythroid burst-forming units, as reported elsewhere. Since the size of this more resistant fraction seems to decrease with stem-cell maturation, the finding implies that this fraction is a primitive subpopulation of the stem-cell compartment. The mean lethal dose (D0), however, of this less sensitive SCID CFU-S is much less than the D0 of regular CFU-S in normal littermates. Spleen colonies produced by SCID bone marrow were relatively small and abortive. The size of these colonies decreased nearly exponentially with increasing doses of radiation. These colonies were believed to be produced by this less sensitive fraction of the stem cells, which carried residual injuries. The colonies produced by the sensitive fraction have disappeared, being killed by a relatively low dose of radiation. This observation might account for the high lymphomagenesis arising from primitive hemopoietic stem cells in SCID mice, because the smallness of the colonies suggests that there is unrepaired or misrepaired damage. Furthermore, this less sensitive fraction might be a source of the "leaky" change of T and B cells, possibly due to the induction of an equivocal repair system which appears in the later stages of life in the SCID mice.

The same exhaustible multilineage precursor produces both myeloid and lymphoid cells as early as 3-4 weeks after marrow transplantation.

Hemopoietic precursors with the ability to differentiate into wide varieties of cell types are considered primitive, as are precursors with long-term repopulating ability. Here we study the populations of marrow precursors from which both myeloid and lymphoid lineages are descended shortly after transplantation. Surprisingly, few or none of these precursors show long-term repopulating ability. Equal portions of a mixture of marrow cells from C57BL/6J (B6) and congenic B6-Hbbd Gpi-1a mice are transplanted into a group of recipients. Three weeks later, highly significant correlations between percentages of B6 type T cells, B cells, granulocytes, and platelets in each recipient indicate that many lymphoid and myeloid cells are descended from common precursors. After 4-6 weeks, most correlations between lymphoid and myeloid cells improve, indicating that most or all differentiated cells are descended from common precursors. The more differentiated myeloid-specific precursors found in spleen colony-forming cell assays apparently fail to contribute significantly to the differentiated myeloid cell populations tested. By using the binomial model, in which variability of the data among the recipients is inversely related to the number of precursors in the mixture, donor precursor concentrations are estimated as approximately 21 per 10(5) marrow cells after 3 weeks, falling 3-fold to 6.6 per 10(5) after 4-6 weeks. This trend continues, with higher correlations, greater variabilities, and donor precursor concentrations of 1.9 per 10(5) marrow cells after 12-14 weeks and 1.4 per 10(5) after 24 weeks. Strong increases in variances between 3 and 12 weeks after transplantation suggest that most or all of the initially active multilineage precursors are exhausted during this time period. The fact that the ability of a hemopoietic stem cell to differentiate into widely disparate lineages is not associated with long-term repopulating ability requires a change in stem cell definitions, since primitive hemopoietic stem cells have traditionally been defined by both these abilities.

In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand.

The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.

Effects of interleukin-3 on murine fetal liver hemopoiesis in utero.

The normal regulation of primitive hemopoietic stem cells (PHSCS) throughout development involves locally produced factors and humoral factors. While considerable information is available on the effects of candidate hemopoietic growth factors (HGFs) on postnatal hemopoietic tissues, little is known about the regulatory events of developing hemopoietic stem cells within fetal microenvironments. Fetal hemopoiesis represents expanding populations and may be under different regulatory control mechanisms. The microinjection of purified interleukin-3 (IL-3), a candidate HGF, into 13-day-old mouse fetuses via the yolk sac, allowed us to evaluate its effects on morphogenetic events and, more specifically, on fetal liver populations using quantitative in vitro clonal assays for hemopoietic precursors. In view of the sensitivity of fetal development during the early organogenetic period, considerable care was taken to identify the stress effects of the surgical laparotomy and the microinjection procedure. Control studies, required to distinguish stress effects of surgical laparotomy and microinjection, clearly revealed that the fetal liver is a sensitive organ responding with limited tissue disorganization, reduced cellularity and erythropoietic activity, as monitored 24 h after experimental intervention. The microinjection of 15 units of IL-3 promoted a significant expansion of depleted liver hemopoietic-cell populations and had stimulatory effects on the distribution of connective tissue mast cells and absolute cell numbers, including hemopoietic precursors (erythroid, granulocyte, macrophage, megakaryocyte), compared to controls. These studies suggest that (1) fetal liver hemopoiesis is selectively sensitive to maternal stress but has an effective regenerative capacity to maintain essential hemopoiesis in utero, and (2) fetal hemopoietic cells require an ability to respond to IL-3 early in fetal development.

HAEMOPOIESIS: A LOTTERY OR GENOMIC DETERMINISM?

British Journal of HaematologyVolume 79, Issue 3 p. 528-529 Free Access HAEMOPOIESIS: A LOTTERY OR GENOMIC DETERMINISM? J. P. Novak, J. P. Novak Institut de Recherche d'Hydro-Québec (IREQ), Varennes, Québec, Canada J3X 1S1Search for more papers by this authorC. C. Stewart, C. C. Stewart Roswell Park Cancer Institute, Buffalo, New York, U.S.A.Search for more papers by this author J. P. Novak, J. P. Novak Institut de Recherche d'Hydro-Québec (IREQ), Varennes, Québec, Canada J3X 1S1Search for more papers by this authorC. C. Stewart, C. C. Stewart Roswell Park Cancer Institute, Buffalo, New York, U.S.A.Search for more papers by this author First published: November 1991 https://doi.org/10.1111/j.1365-2141.1991.tb08071.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES Brown, G., Jones, N. A., Bunce, M., Lord, J. M., Owen, P. J. & Patton, W. N. (1991) Haemopoiesis: a lottery or genomic determinism British Journal of Haematology, 79, 527– 528. Nakahata, T., Gross, A. J. & Ogawa, M. (1982) A stochastic model of self-renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture. Journal of Cellular Physiology, 113, 455– 458. Novak, J. P. & Stewart, C. C. (1991) Stochastic versus deterministic in haemopoiesis: what is what British Journal of Haematology, 78, 149– 154. Noval, J. P. & Stewart, C. C. (1992) A possible interpretation of some colony forming assays: ‘developmental tree’ model. Submitted to Cell and Tissue Kinetics; copies of manuscript available. Volume79, Issue3November 1991Pages 528-529 ReferencesRelatedInformation

Leukemia initiated by hemopoietic stem cells expressing the v-abl oncogene.

We report a mouse model with which to study leukemogenesis initiated by a specific genetic change introduced into a primary lymphoid-myeloid pluripotent stem cell. Fetal liver hemopoietic cells were infected with a high titer of helper-free Abelson murine leukemia virus (A-MuLV) and were used to reconstitute lethally irradiated mice. Two weeks later, progenies of a single primitive hemopoietic stem cell carrying a specifically integrated A-MuLV proviral DNA could be detected in both colony-forming units in spleen and myeloid colony-forming cells in the bone marrow. Beginning at 3 weeks after transplantation, the recipients developed elevated leukocyte counts, splenomegaly, and increase of blast cells in the peripheral blood. Multiple clones of A-MuLV-infected cells were infused into each recipient. However, in the same animal, DNA extracted from various affected organs and from factor-independent lymphoid and myeloid immortalized cells all contained an identical, specifically integrated proviral genome. The A-MuLV-infected stem cells differentiated into various lineages of hemopoietic cells. Our data show that the expression of the v-abl oncogene in a primary lymphoid-myeloid hemopoietic stem cell directly initiates leukemogenesis by stimulating factor-independent growth. The monoclonal-type disease development seen in these animals may require the occurrence of an additional genetic event.

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.

Comparative Effects of Busulfan, Cytosine Arabinoside and Adriamycin on Different Maturation Stages of Normal Human Bone Marrow Cells

Leukemic blast progenitors in acute myeloblastic leukemia (AML) undergo terminal divisions and/or self-renewal, which can be studied by the methylcellulose culture method and suspension culture, respectively. Using these methods, we have shown that busulfan (BU) was more effective against the self-renewal of blast progenitors than against the terminal divisions. As reported previously, cytosine arabinoside (Ara-C) is more effective against self-renewal and adriamycin (ADR) is more effective against terminal divisions. To determine the reason why antileukemic drugs show different sensitivity against self-renewal and terminal divisions, we studied the effects of these drugs on normal human bone marrow cells. We have shown that BU and Ara-C suppressed the colony formation induced by interleukin-3 (IL-3) more effectively than that by granulocyte colony-stimulating factor (G-CSF). On the other hand, ADR suppressed the colonies induced by G-CSF more effectively. In normal hemopoiesis, IL-3 has been shown to stimulate the growth of more primitive hemopoietic stem cells, while G-CSF has been documented to stimulate the growth of granulopoietic committed precursors at a more differentiated stage. Therefore, BU and Ara-C are considered to be more effective against the immature cells in normal bone marrow and ADR is effective against the more differentiated cells. We suppose that the different differentiation stage of target cells may explain the difference of the effects on self-renewal and terminal divisions between BU, Ara-C and ADR.

Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention.

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.

Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells.

The in vitro production of eosinophils from committed progenitor cells is influenced by interleukin (IL)-5 (eosinophil differentiation factor) and to a lesser extent by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In primary suspension cultures of marrow cells taken from eosinophilic mice, IL-3 induced a modest stimulation of eosinophil production compared to IL-5. In contrast, IL-3 was sevenfold more effective than IL-5 in generating eosinophil progenitors (eosinophil colony-forming units (CFU-eo] from more primitive precursors present in the marrow of normal mice. Pre-incubation of marrow cells in suspension culture with IL-3, but not IL-5, increased the recovery of myeloid precursors responsive to G-CSF, GM-CSF, CSF-1, or IL-3 two- to fourfold while eosinophil progenitor cells responsive to IL-5 were increased by more than 70-fold. Similarly, pre-incubation of bone marrow cells under clonal conditions with IL-3, but not IL-5, resulted in a more than 50 fold increase in CFU-eo responsive to IL-5 over input values. Bone marrow from mice pre-treated with 5-fluorouracil is greatly depleted of progenitor cells directly responsive to IL-3 or IL-5. IL-1 which synergistically interacts with various CSF species to confer a clonogenic response by primitive stem cells present in 5-fluorouracil-treated marrow also failed to stimulate eosinophil production. A marked synergism was observed when IL-1 and IL-3 were combined in the suspension pre-culture phase with a more than sixfold recovery of CFU-eo than induced by either factor alone. Furthermore, pre-culture of 5-fluorouracil-treated marrow cells with a combination of IL-1 and IL-3 resulted in a more than 260-fold increase of CFU-eo over input numbers. These data suggest that the concatenate action of IL-1, IL-3, and IL-5 is an absolute requirement for the in vitro generation of eosinophils from primitive hemopoietic stem cells.

Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/W v mice

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.

Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors.

We report identification of a unique class of human hemopoietic colony-forming cells with extensive ability to generate progenitors for secondary colonies. Mononuclear cells isolated from human umbilical cord blood formed colonies consisting of 40-500 blast cells after 25 d of incubation in methylcellulose culture in the presence of erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes. Replating of these blast cell colonies revealed that 100% of the primary colonies had the ability to generate secondary colonies, including multipotential colonies. These colonies could be distinguished from other hemopoietic colonies in situ by the complete absence of signs of terminal differentiation. Replating of granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies, consisting of an average of 2 x 10(4) cells, revealed less capacity for secondary colony formation. This human blast cell colony assay may provide a method for quantitation of more primitive hemopoietic stem cells than progenitors for GEMM colonies (CFU-GEMM) in man.

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.