Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status.

The most primitive hematopoietic stem cells capable of longterm reconstitution of the entire hematopoietic system following transplantation are characterized by their ability to exclude both Rhodamine 123 and Hoechst 33342 dyes (Rh/Ho(dull)), and are an appropriate target population for the determination of stem cell ultrastructure. We have used a fluorescence-activated cell sorter to enrich to near purity these rare, highly quiescent cells. Analysis of the in vitro growth characteristics of Rh/Ho(dull) cells demonstrated an obligatory requirement for multiple growth factors, with 62% of the sorted population having the capacity to form colonies in the presence of CSF-1 + IL-1alpha + IL-3 + SCF. The Rh/Ho(dull) cells were small, with profiles having a mean diameter of 4.6 microm. Ultrastructural examination showed numerous ribosomes and several mitochondria in the thin rim of cytoplasm surrounding the nucleus, with other cytoplasmic organelles revealed in serial sections. The cells were generally homogeneous in appearance apart from the nucleus, which had an irregular shape with a single deep indentation. The heterochromatin around the margin was distinctly more pronounced in about 50% of nuclei. The findings provide a basis for studying the structural changes that occur with progressive differentiation of early hematopoietic cells.

Radiation response of mouse lymphoid and myeloid cell lines. Part II. Apoptotic death is shown by all lines examined.

The mode of death induced by gamma-irradiation in a panel of 10 mouse lymphoid or myeloid cell lines was examined. Four of these lines were known to lose viability (membrane integrity) rapidly after irradiation, whilst the others were known to lose viability considerably more slowly. However, based on the criteria of morphology and DNA degradation pattern, all 10 lines showed apoptotic death. The occurrence of apoptosis after irradiation in rapid-dying lymphoid cell lines was consistent with published results, whilst the demonstration of apoptosis in slow-dying lines was unexpected. Cells of the slow-dying lymphoid lines underwent one or more mitoses prior to death, a feature also reported for fibroblastoid cell lines. However, the occurrence of radiation-induced necrosis in fibroblasts suggests that the pathways leading to 'mitotic death' differ between fibroblastoid and lymphoid cell lines.

Compensatory mechanisms in platelet production: lack of a paracrine response in W/Wv mice treated with 5-fluorouracil.

W/Wv mice maintain normal platelet levels despite having a reduced functional stem cell pool, indicating that platelet production in these mice is compensated by altered megakaryocytopoiesis. In this study the effect of 5-fluorouracil (5-FU) treatment on platelet production in W/Wv mice and their congenic normal littermates was assessed. Recovery of circulating platelet levels occurred 11 days after 5-FU administration in W/Wv mice and subsequently did not increase above control values. In contrast, normal littermates showed an increased platelet count by day 8 and significant thrombocytosis between days 11 and 14. Investigation of bone marrow megakaryocytopoiesis in W/Wv mice showed there was no recovery in the number of megakaryocyte progenitors (CFU-Meg) per femur between days 3 and 5, but control values were reached by day 10. In addition, by day 8 the number of mature megakaryocytes per unit volume of bone marrow in these mice had not returned to control values, although the megakaryocytes were of an increased size. In comparison, the number of CFU-Meg per femur in normal mice treated with 5-FU began to recover after day 3, returned to control values by day 8 and increased to supranormal levels by day 14. Bone marrow megakaryocyte concentration was increased 2-fold over the control by day 8 and an increase in mean megakaryocyte size was also observed. The data suggest that platelet production in mice is dependent on the rate of establishment of both the progenitor cell and megakaryocyte pools. The inability of W/Wv mice to enhance and accelerate progenitor cell levels led to a reduced bone marrow response and failure to produce a marked thrombocytosis.

Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Characterization of endothelial cells in murine long-term marrow culture. Implication for hemopoietic regulation.

Establishing the presence of various cell types in long-term marrow culture (LTMC) has an important bearing on understanding the regulation of stromal cell-related hemopoiesis. Controversy has surrounded the identity of the very large cells in murine LTMC; they have been given a variety of designations, including blanket cells. Using dual immunogold labeling with a recently derived monoclonal antibody, H513E3, and anti-human factor VIII antibodies, we have conclusively located endothelial cells in LTMC. Endothelial cells are relatively large, with thinly spread cytoplasm, and they overlie macrophages, granulocytes, and other less differentiated developing hemopoietic cells. Previously, demonstration of endothelial cells in LTMC had been difficult due to lack of specific markers in the mouse. We have demonstrated that LTMC endothelial cells have the exact location and ultrastructural characteristics as the previously described blanket cells. We propose that previous designations should not be continued and that these cells should be referred to as endothelial cells. The known functions of endothelial cells now become relevant to the understanding of stromal regulation of hemopoiesis.

Megakaryocyte maturation in long-term marrow culture.

Evidence has been sought for megakaryocyte maturation in long-term cultures of mouse bone marrow. Cultures up to 14 weeks of age were examined for the presence of megakaryocytes with processes, that is, resembling the morphological appearance seen in vivo prior to platelet liberation. Such cells were found floating just above the adherent stromal layer using low magnification phase contrast microscopy. It was rare to observe as many as 20 of these cells per 25-cm2 flask. At higher magnification, processes were seen to be attenuated with constrictions at intervals along their length. Time-lapse photography was used to follow the development and behavior of the processes. Direct evidence of rupture was very rare; generally the megakaryocytes retracted their processes within 48 h. Careful searching of cultures occasionally revealed the presence of several process fragments, and sometimes individual platelets were found. Ultrastructurally, the processes were seen to contain organelles that are usually associated with platelets. The observations applied to both Dexter and Whitlock-Witte cultures. It is concluded that maturation of megakaryocytes occurs in long-term marrow culture to the point where platelet release appears imminent. Final rupture is rare and may require shearing forces, which in vivo would be provided by blood flow.

A mouse endothelial cell-specific monoclonal antibody: its reactivity with LTMC endothelium.

The binding of a marrow cell-related monoclonal antibody (H513E3 MAb) has been investigated in long-term marrow cultures (LTMC) and in vivo. Immunogold labeling and electron microscopy revealed that this antibody labeled an endothelial-like cell. Cross-reaction of anti-human Factor VIII confirmed endothelial specificity of the H513E3 MAb. In addition, vessel endothelium (vena cava, aorta, and marrow) exhibited binding of the antibody. This antibody provides a unique tool to study the cellular architecture of LTMC and implicates endothelium as an important component of LTMC. The function of the endothelial cell-specific surface antigen is unknown, although preferential labeling of the upper surface of endothelial cells suggests that it may play a role in cell communication, particularly with the floating population. The H513E3 MAb reacts with an external membrane antigen, a property that makes this antibody particularly useful for fluorescences-activated sorting of endothelial cells.

Use of bone marrow somatic cell hybrid lines to generate monoclonal antibodies specifically reactive with rare marrow cells.

The technique of somatic cell hybridization has been applied to obtain new sources of immunogen for monoclonal antibody production. A marrow population enriched for rare and undifferentiated cells was hybridized with A9 cells. Mature cells were depleted by using mice at 8 days following 5-fluorouracil treatment and by using a sorting procedure. Selection of hybrids was in medium containing hypoxanthine, aminopterin, and thymidine (HAT). Chromosome analysis was used to verify that clones contained hybrid cells. An example of an antibody (H513E3) obtained by immunizing with a hybrid cell line (H5Scl1.4.4) is presented. The H513E3 antibody showed low reactivity with normal marrow (0%-3%), and hemopoietic cell lines of various types exhibited no cross-reaction. However, about 10% of cells from the adherent stromal layer of long-term marrow cultures reacted with the H513E3 antibody. A specific cell type was labeled that appeared to have endothelial-like morphology when observed with immunogold labeling and electron microscopy. The hybridization technique allows a cell of a particular differentiation lineage to be conserved, wholly or partially, in a cloned form, thus overcoming the heterogeneity of a normal marrow population.

Megakaryocyte fragments and the microtubule coil.

We have examined megakaryocyte process fragments that migrate out of bone marrow explants after a short period of incubation and assume a beaded form, consisting of 2 or more putative platelets. The fragmentation appears to occur in vivo and supports the proposal that platelet liberation does not always occur in a sequential manner from the distal ends of megakaryocyte processes. Transmission electron microscopy revealed that microtubules were generally oriented longitudinally in the process fragments. Rarely, a microtubule coil was found in a terminally located putative platelet. The observations favour the view that the marginal coil of microtubules, which is a characteristic of circulating platelets, does not usually form until after platelets have been liberated.

Characterization of megakaryocyte spleen colony-forming units by response to 5-fluorouracil and by unit gravity sedimentation.

Properties of megakaryocyte progenitor cells in mouse bone marrow have been examined using an in vivo assay system. Perturbation with 5-fluorouracil (5-FU) and separation by unit gravity sedimentation was used to characterize the cells. Bone marrow was assayed for the presence of megakaryocyte colony-forming cells (MK CFU-S) by transplantation into lethally irradiated mice and examining spleen sections 10 days later. Donor mice were untreated or injected intravenously with 5-FU (150 mg/kg), 1 (FU-1) or 7 (FU-7) days beforehand. There was a lack of correlation between the numbers of MK CFU-S and cells giving rise to macroscopic spleen surface colonies (CFU-S10). The sedimentation profile of MK CFU-S in normal marrow was similar (modal velocity 4.16 +/- 0.05 mm/hr) to that of CFU-S10. In FU-1 marrow, MK CFU-S exhibited a bimodal sedimentation profile, with peaks at 3.26 +/- 0.06 mm/hr and 4.53 +/- 0.07 mm/hr. The marrow content of CFU-S10 was reduced to 5% of normal, while MK CFU-S numbers were only reduced to 60%. In FU-7 marrow, the sedimentation profile of MK CFU-S (modal velocity 4.86 +/- 0.16 mm/hr) differed from that of CFU-S10 (5.5 +/- 0.16 mm/hr). It was concluded MK CFU-S and CFU-S10 are different entities. The MK colonies formed from FU-1 marrow contained on average 3.8-fold more cells than those formed from normal marrow. The enhanced megakaryocyte production may be accounted for on the basis of the generation-age model for cell proliferation. It is proposed that MK CFU-S are a heterogeneous population with regard to proliferation potential and that the FU-1 marrow contains cells that survive 5-FU and have a high proliferative potential. These cells may be equivalent among megakaryocytic progenitors to the high proliferative potential colony-forming cells of the granulocyte/macrophage series. They may be responsible for the enhanced megakaryocytopoiesis seen in the marrow of mice 7 days after the injection of 5-FU.

In vitro production of CFU-S and cells with erythropoiesis repopulating ability by 5-fluorouracil treated mouse bone marrow.

Mouse bone marrow, obtained from donors three days after treatment with 5-fluorouracil, had a very low ability to form macroscopic spleen colonies in irradiated mice at 10 days after transplantation of the cells (CFU-S10); such marrow also had no detectable erythropoiesis repopulating ability but did have near normal marrow repopulating ability and spleen megakaryocyte repopulating ability. Incubation of this marrow in vitro for 7 days with medium containing growth factor preparations (a) pregnant mouse uterus extract plus human spleen conditioned medium or (b) mouse spleen conditioned medium, resulted in marked increases in CFU-S10 and in cells with erythropoietic repopulating ability together with maintenance of cells with marrow repopulating ability. These responses were not observed in cultures with control medium alone. Spleen megakaryocyte repopulating ability was also maintained in the presence of the factor preparations.

Fate of senescent megakaryocytes in the bone marrow.

Degenerating senescent megakaryocytes have been identified in mouse bone marrow by light and electron microscopy. The ultrastructural changes which occur as degeneration proceeds are characteristic of death by apoptosis, although most cells appear to round up rather than undergo fragmentation. A hitherto unreported finding in degenerating cells was the presence of bundles of approximately 7 nm diameter parallel filaments in nuclei and membrane-bound nuclear fragments. Structurally, they resembled bundles of filaments induced in nuclei with dimethyl sulphoxide and identified as actin. Often a bundle appeared to terminate at the inner membrane. In the cytoplasm the presence of microtubules and centrioles indicates that not all the latter organelles are lost by the megakaryocyte during platelet release. Degenerating senescent megakaryocytes are rare in the marrow of normal mice but increase in frequency during 5-fluorouracil stimulated thrombocytosis. The dying cells are eventually phagocytosed by macrophages, a process that can occur extravascularly, showing that entry of senescent megakaryocytes into the circulation is not necessary for their disposal.

Time-lapse cinemicrography and scanning electron microscopy of platelet formation by megakaryocytes.

The surface architecture of megakaryocytes undergoing platelet formation in vitro has been examined by time-lapse cinemicrography and scanning electron microscopy. Fragments of mouse bone marrow were placed in culture medium and incubated at 37 degrees C. After several hours mature megakaryocytes migrated out of the marrow and some underwent shape changes so that they eventually appeared as a relatively small central body, housing the nucleus, from which emerged a number of thin processes which resembled platelet chains. Scanning electron microscopy showed that initially the megakaryocyte surface was ruffled but with development of processes it became smoother. Circumferential folds of small amplitude were found on the surface of developing constrictions which separated putative platelets. It is thought they may be associated with the mechanism of extension, but could have a role in establishing the topography of membrane components. Rupture of the chains and release of platelets was not observed; this permits the number of putative platelets formed by individual megakaryocytes to be determined. The putative platelets exhibited features common to circulating platelets when exposed to a glass surface including the development of pseudopodia and, eventually, flattening on to the surface.

The demarcation membrane system of the megakaryocyte: a misnomer?

The concept that the demarcation membrane system delineates platelets within the cytoplasm of megakaryocytes has been examined. In short-term culture of mouse bone marrow, mature megakaryocytes extended long, attenuated processes that were found by electron microscopy to have a limited amount of invaginated membrane. When such megakaryocytes were exposed to microtubule depolymerizing agents, the attenuated processes retracted, became thicker, and an extensive demarcation membrane reappeared. It is suggested from the results that the demarcation membrane system functions to provide a membrane reserve that undergoes evagination during the formation of attenuated processes and thereby envelops putative platelets, rather than to demarcate platelets in the maturing megakaryocyte. The term "invaginated membrane system" is considered more appropriate than "demarcation membrane system."

The organization of hemopoietic tissue as inferred from the effects of 5-fluorouracil.

Mouse bone marrow obtained one day after injection of 5-fluorouracil (FU) had a markedly diminished content of spleen colony forming units (CFUs) but retained its capacity to repopulate the marrow granulocyte-macrophage colony forming cell (GM-CFC) and CFUs compartments of 850 R irradiated hosts and had only a slightly reduced platelet repopulating ability (PRA). A significant correlation (r = 0.94, P less than 0.001) was observed between the content of high proliferative potential granulocyte-macrophage progenitor (HPP-GM-CFC) and the platelet and marrow GM-CFC repopulating abilities of bone marrow cell suspensions. Spleens of irradiated mice, injected with marrow from donors treated with FU between 1 and 7 days before showed an increase in colony numbers with time of sampling between 8-13 days after transplantation. In contrast, the colony counts observed in mice injected with normal bone marrow remained constant over that time interval. The colonies derived from bone marrow of FU treated mice grew faster than those from bone marrow of normal mice. Spleens obtained from irradiated mice, 10 days after injection of bone marrow derived from donors treated with FU 1 or 3 days before, showed only a few macroscopic surface colonies but when sectioned were found to contain large numbers of microscopic colonies, 80% of which were megakaryocytic. The results are interpreted on the basis of a clonal succession model of hemopoiesis with stem cells of varying proliferative potential and proliferation rates increasing as capacity for cell production decreases.

Aspects of platelet formation and release.

The surface architecture and location of megakaryocytes in the extravascular compartment of mouse bone marrow are considered. The finger-like cytoplasmic processes that extend into sinuses pass through apertures, approximately 3.0 millimicron in diameter, occurring singly or in groups. The cytoplasmic processes enlarge on first entering a sinus and form villi that seem to anchor them to endothelium. The villi consist largely of microfilaments. The cytoplasmic processes extend, attenuate, and undergo irregular constriction along their lengths. The considerations demarcate modicums of cytoplasm of platelet size one from another that are released when they break. Extravascular platelet release may also occur, and evidence is presented that points to the likelihood of the platelet's being engulfed and phagocytosed by macrophages before reaching the microcirculation.

Extravascular phagocytosis of proerythrocytes following 5-fluorouracil.

Amongst the perturbations induced in mouse bone marrow by 5-fluorouracil are the retention and phagocytosis of proerythrocytes within the extravascular compartment. Reasons for the latter response have been sought using electron microscopy. Proerythrocytes undergo normal maturation after 5-fluorouracil, judged by loss of mitochondria and vesicles, but fail generally to attain a biconcave shape. Most proerythrocytes have lost their organelles before phagocytosis occurs. Quantitative light microscopy shows a rise in the number of proerythrocytes 1 d after 5-fluorouracil which is attributable to maturation of orthochromatic erythroblasts. Thereafter the population size declines concomitantly with increased engulfment and phagocytosis by macrophages, activity which is maximal on days 4-5. Extravascular loss of proerythrocytes is also observed when marrow is rendered hypocellular by x-irradiation, or hydroxyurea. It is suggested that excessive maturation of proerythrocytes in the extravascular compartment can lead to their phagocytosis.

Enhanced megakaryocyte repopulating ability of stem cells surviving 5-fluorouracil treatment.

The effect of 5-fluorouracil treatment of donor mice on the capacity of transplanted bone marrow to produce megakaryocytes in the spleens of lethally irradiated recipients has been examined. At both 10 and 13 days after transplantation, the spleens of recipients of 5-fluorouracil treated bone marrow had significantly more megakarocytes per unit area of spleen section than recipients injected with an equivalent number of spleen colony forming units from normal bone marrow. It is suggested that such treatment may provide a sensitive in vivo system for the investigation of endogenous factors influencing megakaryocyte progenitor proliferation. The results are consistent with the concept of stem cells being heterogeneous with respect to self-renewal capacity.

Effects of 5-fluorouracil on mouse bone marrow.

5-Fluorouracil (150 mg/kg) was injected intravenously into 11-week-old male mice. Its effects on marrow structure over the following 9 d were assessed using light microscopic examination of semi-thin transverse sections of decalcified humeri. The extravascular compartment became markedly depleted of cells and decreased in size, reaching a minimum on day 5 post-treatment. Proliferating haematopoietic cells disappeared within 2 d. Post-replicative erythrocytic cells underwent extra-vascular phagocytosis by resident macrophages, whilst mature granulocytes continued to pass into the circulation. Repopulation was underway by day 6, and re-expansion of the extravascular compartment by day 7 when megakaryocytes, of larger than normal average size, occupied most of the extravascular compartment. Megakaryocyte dominance gave way over days 8 and 9 as other haematopoietic cells proliferated and the extravascular compartment continued to enlarge. The significance of the results is discussed in relation to marrow micro-circulation, egress of haematopoietic cells into the circulation, and cell proliferation in the marrow. Endotoxin, administered 4 h after 5-fluorouracil, accelerated depletion of the marrow and recovery of haematopoiesis by about 1 d.