Erythroblastic Differentiation of Stem Cells in Hemopoietic Colonies

An early phase of cellular replicationprecedes the initiation of erythropoiesis in hemopoietic spleen colonies ofmice receiving a supralethal split-doseof irradiation, first with the leg shieldedand then followed by leg irradiation3 hr later. Previous studies indicatedthat this proliferative phase representsthe repopulation of a depleted, endogenous stem cell compartment. Tolabel replicating stem cells, tritiatedthymidine (³HTdR) was injected intraperitoneally in mice on days 1-4 afterirradiation. Touch preparations andautoradiograms of splenic colony cellswere examined with light and electron microscopy from 2 to 4 days afterirradiation and 5-8 days later, whenerythroblasts and other differentiatedcells first appeared. Replicating mononuclear cells that were pulse labeledduring the early proliferative phase ofstem cell renewal resembled medium- to large-sized leptochromatic lymphocytes. Electron microscopic examination of autoradiograms demonstrateda labeled undifferentiated cell with athin rim of nuclear heterochromatin.Sequential studies showed that the injection of ³HTdR on days 1-4, whenonly the mononuclear cells were present, resulted in labeled erythroblastsand other differentiated cells on days5 and 6. These results confirm thepresence of an early proliferative phasein endogenous splenic hemopoieticcolonies and strongly suggest thatmononuclear cells, replicating duringthis period of self-renewal of the stemcell compartment, transform to erythroblasts and other cells.

Submitted on May 13, 1972 Revised on July 3, 1972 Accepted on July 9, 1972     

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.     

Distribution of 51Cr labeled leukemia cells in mice: comparison with representative normal cells

Cells of two transplantable leukemias of mice, one myeloid and one lymphoid, were labeled with 51Cr in order to follow their distribution in hemopoietic and parenchymatous organs and blood of syngeneic recipients. Distribution of myeloid leukemia cells was compared with that of regenerating bone marrow cells and normal spleen cells. The organ distribution of myeloid leukemia cells was essentially different from that of cells of regenerating bone marrow, and both were different from that of normal spleen cells. Cells of lymphoid leukemia, which are presumably of B-lymphocyte origin, were compared with a B-lymphocyte enriched population, obtained from the lymph nodes of so-called TIR mice (thymectomized, irradiated, and reconstituted with syngeneic bone marrow), and with spleen cells of normal mice. The three patterns of organ distribution were different. It is concluded that the two leukemias studied each have a specific and characteristic distribution.     

Effect of two cyclophosphamide derivatives on hemopoietic progenitor cells and pluripotential stem cells

The studies described herein were undertaken to help define the effects of certain cyclophosphamide derivatives that have been used for selective removal of leukemic cells from marrow samples used for autologous transplantation. We have tested the effect of 4-HC and another cyclophosphamide congener, ASTA-Z 7557, on pluripotent stem cells (CFU-S) and committed progenitor cells (CFU-GM) in mice. The CFU-S were evaluated by the spleen colony assay at eight days and 12 days after transplant. The eight-day colonies are transient in nature, rapidly growing, mainly erythroid, and lack pluripotential precursors. The 12-day colonies are believed to provide a measure of hemopoietic stem cells as they slowly grow and do contain primitive precursors. Our data show that at the maximum dose levels tested, both drugs caused a 100% loss of CFU-GM and about 80%-95% inhibition of early transient CFU-S. In contrast, about 70% of the pluripotent 12-day CFU-S were spared. These data appear to explain the hemopoietic recovery seen in man after transplantation with marrow cells treated with 4-HC despite their relative absence of hemopoietic progenitor cells.     

The CD34 hemopoietic progenitor cell associated antigen: biology and clinical applications.

CD34, which was first detected in hemopoietic and lymphopoietic progenitors, is a heavily glycosylated Type I transmembrane protein that does not share any significant similarity with other transmembrane proteins. Its functions are still unknown. Several monoclonal antibodies were raised against CD34, and at least 4 different epitopes could be recognized. CD34 expression is confined to a few cell lines, to 1-4% of adult bone marrow mononuclear cells (including marrow-repopulating cells, all multipotent and committed myeloid progenitors, B and T lymphoid precursors, osteoclast precursors, and most likely the precursors for stromal cells), and to less than 1% of peripheral blood mononuclear cells. In non-lymphohemopoietic tissues its expression is confined to endothelial cells and to some cells of the skin. In malignancies, CD34 expression is not fully elucidated. Immature hemolymphopoietic malignancies (namely acute leukemias) and the blast cells of chronic myeloid leukemia are frequently positive. Chronic lymphoproliferative disorders and lymphomas are negative. Among other tumors, only vascular derived tumors are positive. Clinical applications of CD34+ cells include autologous transplantation of putative CD34+ stem cells isolated by positive selection from the bone marrow, and transplantation of autologous peripheral blood stem cells, using the proportion and number of CD34+ cells as a guideline for the harvesting procedure.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

Summary The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4–10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

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.     

Suppressor T-cell clones derived from pluripotent stem cells (CFU-GEMM) of a patient with hodgkin's lymphoma

Summary Pluripotent stem cells (CFU-GEMM) give rise to multilineage hemopoietic colonies in culture. The cellular composition revealed that mixed colonies contain cells of different myeloid lineages and mononuclear cells with T-cell surface antigens. T-lymphocytes of primary colonies, replated secondary and tertiary colonies from a patient with Hodgkin's Lymphoma were identified by their reaction with the monoclonal antibody OKT 8. Evidence for a common progenitor of myeloid and lymphoid cells is provided by analysis of individual secondary and tertiary colonies using OKT 3, OKT 4, OKT 8, VIM-D 5, and Ig M + D antibodies for each individual colony. Primary mixed, replated secondary and tertiary colonies revealed OKT 8 positive cells. No reaction with OKT 3, OKT 4, VIM-D 5, OR Ig M+D was observed.Supported by Deutsche Forschungsgemeinschaft     

Murine spleen culture: homing of hemopoietic progenitor cells to spleen is not mediated by a similar mechanism to that in marrow

We have previously shown that in long-term bone marrow cultures (LTMC) the specific recognition and binding of hemopoietic stem cells to stroma, which we call "homing," is mediated by a recognition mechanism involving a surface membrane lectin with galactosyl and mannosyl specificities. Subsequent in vivo studies in lethally irradiated mice confirmed that homing to the marrow similarly involves a galactosyl- and mannosyl-specific recognition mechanism. However, these in vivo studies suggested that homing of hemopoietic progenitor cells to spleen was based upon a different molecular recognition mechanism. In the present study splenic homing was investigated in a cell culture system composed of an adherent layer of splenic stromal cells inoculated with stroma-free stem cells from the supernate of LTMC. In this system, splenic stroma supported proliferation and differentiation of hemopoietic precursors for a few weeks. When stem cells were added to the cultures in the presence or absence of inhibitory concentrations of neoglycoprotein reagents specific for galactosyl, mannosyl, or fucosyl lectins, the pattern of production of total cells, pluripotential stem cells (CFU-S), and granulocyte-macrophage committed progenitors (CFU-GM) remained the same. These data support our in vivo observations that homing of stem cells to splenic stroma is not mediated by a surface lectin with galactosyl and mannosyl specificities as it is in bone marrow, but rather by a different molecular mechanism.     

Retrovirus-mediated gene transfer to purified hemopoietic stem cells with long-term lympho-myelopoietic repopulating ability.

Despite recent advances in marrow stem cell purification, controversy about the nature and heterogeneity of cells with the potential for long-term repopulation of lymphoid and myeloid tissues remains. Essential to the resolution of these questions is the use of strategies to track the progeny produced in vivo from individual hemopoietic stem cells in purified populations. We have used a procedure for obtaining highly enriched populations of stem cells with competitive repopulating ability from male mice (pretreated with 5-fluorouracil), and in this paper we present the results of studies in which small numbers (150-2000) of these cells were exposed to supernatant containing a helper-free recombinant retrovirus carrying the neomycin-resistance gene and then were transplanted together with 2 x 10(5) "compromised" female marrow cells into irradiated female recipients. Male cells--i.e., progeny of purified stem cells--were found in one or more of the tissues examined (peripheral blood, marrow, spleen, and thymus) in 28 of 28 mice evaluated at various times between 35 and 196 days after transplantation. In 20 of these mice (71%), the neomycin-resistance gene was also detected, although not always at a level that correlated with the proportion of male cells. Analysis of spleen colonies (day 12) generated in secondary recipients confirmed that viral integration was confined to male repopulating cells. In three mice direct evidence of a common clone in both lymphoid and myeloid tissues was also obtained. These results show the feasibility of retrovirus-mediated gene transfer to highly purified populations of lympho-myelopoietic stem cells with long-term (6 months) repopulating potential by using a supernatant infection protocol. This approach should facilitate further analysis of hemopoietic stem cell control in vivo and find future applications in the evolving use of bone marrow transplantation for hemopoietic rescue and gene therapy.     

Culture studies of human pluripotent hemopoietic progenitors

Conclusions Culture conditions are described that promote the growth of human pluripotent hemopoietic progenitors and facilitate their quantitation. These primitive cells form mixed colonies that may contain all elements of myeloid differentiation, including granulocytes, erythroblasts, megakaryocytes, and macrophages. Some mixed colonies contain, in addition to mature progeny, early progenitors that can be identified by their ability to form secondary hemopoietic colonies. The production of secondary mixed hemopoietic colonies by cells present in redispersed primary mixed hemopoietic colonies supports the view that some CFU-GEMM may self-replicate in culture, thus fulfilling one of the major operational requirements for pluripotent hemopoietic stem cells. Assessment of the proliferative state of CFU-GEMM under steady state conditions and in various clinical disorders suggests that the proliferative activity may reflect conditions that are associated with perturbations at the level of pluripotent progenitors.The recently observed increased plating efficiency of approximately 10 mixed colonies per 10⁵ plated mononuclear cells will facilitate investigation of regulatory events at the level of pluripotent progenitors. With the use of putative stimulators, it might be feasible to modulate the cellular composition of mixed colonies and thus, identify mechanisms that determine the choice of pluripotent cells to self-replicate or to differentiate and mature into cells of specific phenotype.Supported by the Medical Research Council of Canada, University of Toronto and the Banting Research FoundationFellow of the Medical Research Council of Canada and financially assisted by Deutsche Forschungsgemeinschaft     

Paroxysmal nocturnal hemoglobinuria terminating in TdT-positive acute leukemia

A case of paroxysmal nocturnal hemoglobinuria (PNH) which developed terminal transferase (TdT)-positive leukemia 5 years after the diagnosis of PNH was studied. Most of the leukemic cells were suggestively lymphoid by cytochemistry and electron microscopy, and TdT-positive by immunofluorescence studies. The development of acute lymphoblastic leukemia during the course of PNH suggests that in PNH the clonal abnormality may involve lymphoid cells as well as myeloid cells, thus raising the possibility of the disease being a disorder of the pluripotential stem cell.     

Hemopoietic cell dysfunction in murine lupus

Irregularities have appeared through almost the entire detectable range of hemopoiesis, from stem cells to functional mature populations in several models of murine lupus. The documentation of widespread abnormalities in many cell lineages implies the existence of a common, defective ancestor, perhaps the pluripotential hemopoietic stem cell. Of major concern are the microenvironmental pressures that may be driving hemopoiesis to its pathological state. The studies to date have not isolated the hemopoietic components from their cellular surroundings. Hence, the existence of a primary defect in any particular cell compartment is as yet an unanswerable point. Additionally, maternal forces must be considered as environmental factors whose consequences may extend into postnatal life. Also, the possible existence of hemopoietic cell influences on their environs are always present, creating a cellular ecosystem. As new techniques become available for the analysis of these cells, such as long-term cell culturing, more complete pictures of these murine models of autoimmunity will emerge.     

Studies on the effect of deoxyadenosine on deoxycoformycin-treated myeloid and lymphoid stem cells

Adenosine deaminase (ADA) deficiency has been reported in association with severe combined immunodeficiency disease (SCID). The mechanism by which ADA deficiency causes immune dysfunction has been investigated in model systems to which the ADA inhibitor deoxycoformycin (dCf) had been added. Previously, we demonstrated that dCF did not prevent proliferation and differentiation of myeloid and lymphoid stem cells. We have now shown that addition of deoxyadenosine to dCf-containing cultures inhibited proliferation of hemopoietic stem cells. This inhibition was, however, equally effective for both normal myeloid and lymphoid stem cells. These findings suggest that other differences may exist between SCID myeloid and lymphoid stem cells to account for the relative sparing of myelopoiesis in SCID patients.     

Development of thymic and splenic dendritic cell populations from different hemopoietic precursors.

The antigen-presenting dendritic cells (DCs) found in mouse lymphoid tissues are heterogeneous. Several types of DCs have been identified on the basis of the expression of different surface molecules, including CD4, CD8alpha, and DEC-205. Previous studies by the authors showed that the mouse intrathymic lymphoid-restricted precursors (lin(-)c-kit(+)Thy-1(low)CD4(low)) can produce DCs in the thymus and spleen upon intravenous transfer, suggesting a lymphoid origin of these DCs. In the current study, the potential for DC production by the newly identified bone marrow (BM) common lymphoid precursors (CLPs), common myeloid precursors (CMPs), and committed granulocyte and macrophage precursors was examined. It was found that both the lymphoid and the myeloid precursors had the potential to produce DCs. All the different DC populations identified in mouse thymus and spleen could be produced by all these precursor populations. However, CLPs produced predominantly the CD4(-)CD8alpha(+) DCs, whereas CMPs produced similar numbers of CD4(-)CD8alpha(+) and CD4(+)CD8alpha(-) DCs, although at different peak times. On a per cell basis, the CLPs were more potent than the CMPs at DC production, but this may have been compensated for by an excess of CMPs over CLPs in BM. Overall, this study shows that the expression of CD8alpha does not delineate the hemopoietic precursor origin of DCs, and the nature of the early precursors may bias but does not dictate the phenotype of the DC product.     

FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells

The role of caspase-8 and its adaptor Fas-associated death domain (FADD) in lymphocyte apoptosis is well defined, but their functions in other hemopoietic lineages are not clear. We were unable to generate transgenic mice expressing dominant inhibitors of FADD or caspase-8 in hemopoietic cells, possibly because their expression may have precluded production of vital hemopoietic cells. When using a retroviral gene delivery system, fetal liver stem cells expressing a dominant-negative mutant of FADD (FADD-DN) were unable to generate myeloid or lymphoid cells upon transplantation into lethally irradiated mice. However, fetal liver stem cells expressing very low levels of the caspase-8 inhibitor cytokine response modifier A (CrmA) could reconstitute the hemopoietic system. This level of CrmA expression provided some protection against Fas ligand (FasL)-induced apoptosis and promoted accumulation of myeloid cells in the bone marrow, but it did not inhibit mitogen-induced proliferation of B or T lymphocytes. Using an in vitro colony formation assay, we found that fetal liver stem cells expressing FADD-DN, CrmA, or a dominant-negative mutant of caspase-8 could not proliferate in response to cytokine stimulation. These data demonstrate that the enzymatic activity of caspase-8 and its adaptor FADD are required for cytokine-induced proliferation of hemopoietic progenitor cells.     

In vitro differentiation of embryonic stem cells into lymphocyte precursors able to generate T and B lymphocytes in vivo.

Embryonic stem cells can be induced in vitro, by coculture with the stromal line RP.0.10 and a mixture of interleukins 3, 6, and 7, to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+, PgP-1+, c-kit+, Joro75-, B-220-, F4/80-, Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny.     

Cycling of peripheral blood and marrow lymphocytes in cyclic neutropenia.

A 16-month-old male patient with cyclic neutropenia was found to have cyclic fluctuations of monocytes, lymphocytes, platelets, and eosinophils in the peripheral blood. Changes in lymphocyte counts were not obviously related to B, T, or natural killer cells. All classes of immunoglobulins were elevated throughout the cycle. Studies of the marrow morphology revealed remarkable cyclic oscillations of lymphoid as well as myeloid lineage cells. Granulocyte-macrophage progenitors (CFU-c) cycled and were virtually absent 1 wk prior to the neutropenic nadir. The cyclic changes in marrow lymphoid cell numbers were primarily due to changes in numbers of surface immunoglobulin negative (sIg-), cytoplasmic Ig+ (cIg+) pre-B cells. Pre-B cell numbers cycled from normal to extraordinarily elevated values with the same periodicity but reciprocal to the neutrophil cycle. We propose that the primary defect in cyclic neutropenia may either be a periodic failure of an early myeloid differentiation factor or a blunted response of early myeloid precursors to a common hemopoietic growth factor. This may lead to periodic fluctuations in the production or delivery of growth factors (or factor) that influence early stages of differentiation of other hemopoietic cells, including pre-B cells. The essential periodic deficiency is consequently reflected in deficient production of CFU-c accompanied by excessive production or accumulation of pre-B cells (and probably other hemopoietic precursors) in the marrow.     

Characteristics of hemopoietic stem cells determined by single spleen colony transplantation

By using a spleen colony transfer technique and sex chromosome determinations as a cytogenetic marker, we compared the spleen colony-forming cells of bone marrow origin from three strains of mice, i.e., LACA, C57, and (LACA female X C57 male) F1. The proliferation potential of spleen colony-forming cells was found to be heterogeneous in nature. Some of the cells have the essential characteristics of hemopoietic stem cells (Ps-CFUS), i.e., the capacity for sustained proliferation and differentiation into myeloid and lymphoid lineages of cells, whereas others exhibit the capacity to form gross colonies on irradiated recipient spleen which are capable of differentiating different lineages of blood cells, but have lost the ability to reconstitute hemopoiesis in lethally irradiated recipients (Pg-CFUS). The results of studies on spleen colony-forming cells of F1 mice indicate the existence of a subpopulation of primitive precursors of spleen colony-forming cells (Pre-CFUs) which are not able to form spleen colonies on recipient spleens, but, under appropriate stimuli, may assume the properties of Ps-CFUS. Therefore the sequential process of blood cell differentiation may be described as follows: Pre-CFUS----Ps-CFUS----Pg-CFUS----different lineages of hemopoietic progenitors----different lineages of blood cells.     

Mechanisms of compensation of hemolytic anemia in a lactate dehydrogenase mouse mutant

Hemopoiesis was studied in homozygous lactate dehydrogenase (LDH) mutant mice not showing noticeable impairment in viability and fertility but afflicted with a severe hemolytic anemia. In order to investigate the mechanisms of erythropoietic compensation, the numbers of multipotent hemopoietic stem cells (CFU-S), myeloid (GM-CFC), and early and late erythroid progenitors (BFU-E and CFU-E) in femur and spleen were determined, and the total body content of each cell type was computed. While the total CFU-S and GM-CFC numbers showed only slight deviations from normal, the total BFU-E pool was 1.4 and the CFU-E pool 18 times enlarged. No difference in cell cycle status could be detected in these compartments by means of tritiated thymidine (3H-TdR) suicide in vitro. However, splenic erythroblasts of homozygous LDH mutants had a shorter DNA synthesis time and a higher labeling index compared to the wild type mice. It is concluded that the hemolysis is compensated at a lower level of red blood cell count primarily by an increase in the total number of late erythroid progenitors resulting from roughly four extra divisions, and secondarily by an increase in the flux through the recognizable erythroblast compartments, predominantly a space-saving mechanism.