Hemopoietic support capacity of W/WV femurs and tibias.

We evaluated the capacity of "stromal stem cells" of the bones of W/WV mice to effect regeneration of functional "stromal" tissue by implanting femurs and tibias from congenic W/WV and +/+ mice subcutaneously into congenic mice of normal hematologic phenotypes. Eight weeks after implantation, we assayed the hemopoietic progenitor cell contents of the bones. Pluriopotent hemopoietic stem cells (CFUs) were assayed by the spleen colony forming assay in lethally irradiated CAF1 mice. Granulocyte/macrophage progenitor cells (CFUGM) and early erythroid (BFUe) and late erythroid (CFUE) progenitor cells were assayed in agar and methylcellulose semi-solid culture systems, respectively. total cellularity was greater in implanted W/WV femurs and tibias than in +/+ femurs. Although there was substantial variation in the repopulation of W/WV femurs compared to that of +/+ femurs, on the whole, in the W/WV implanted femurs, the numbers of CFUs may have been slightly less and the numbers of maturer progenitor cells slightly greater than they were in the +/+ femurs. All of the progenitor cells assayed were more numerous in the implanted W/WV tibias than in the +/+ tibias. These findings suggest that "stromal stem cells" of W/WV marrow are not defective. In addition, the fact that there were more CFUs in th W/WV that in the +/+ tibias suggests that the CFUs which are native to the implanted bones are not needed to effect reconstitution of normal numbers of CFUs in the implants as all of the CFUs detected in W/WV femurs must have come from the host, for W/WV CFUs do not form surface spleen colonies and, therefore, would not have been counted.

Effects of carrageenan on the mouse hematopoietic system.

We investigated the effects of carrageenans (CAR) on mouse hematopoiesis, one of the many biologic systems affected by these galactan polysaccharides. Mice were injected intravenously with potassium CAR (K+-CAR) or iota CAR (I-CAR) and studied for 7 or 14 days, respectively, thereafter. Treatment with either compound induces anemia, granulocytosis, and early profound thrombocytopenia. Treatment with I-CAR results in an early lymphocytosis, and both compounds induce lymphopenia by 18 h after treatment. Treatment with either CAR compound is associated with an early moderate reduction in the number of nucleated cells and granulocyte/macrophage colony forming cells (CFUGM) per femur. Both compounds induce splenomegaly, and I-CAR treated mice develop hypoplasia of the thymus by 18 h after treatment. The splenomegaly is associated with intense splenic hematopoiesis and an increase in the number of spleen histiocytes; many of the latter are engorged with metachromatically staining material, most likely CAR. There is a sustained increase in the numbers of spleen CFUGM after treatment with either compound; in the case of I-CAR this may be due to proliferation of CFUGM in this organ, perhaps effected by the increased levels of plasma colony stimulating activity. Although it has been suggested that I-CAR is relatively nontoxic, and, therefore, potentially useful for in vivo studies, our observations indicate that it has profound effects on hematopoiesis which must be considered when planning and interpreting in vivo studies using this compound.

Standardization of the Romanowsky staining procedure: an overview.

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.

Antiserum to mouse hematopoietic pluripotent stem cells (CFU-S): further investigation of its functional properties.

Treatment of mouse hematopoietic cells with heterologous antiserum raised against mouse brain markedly reduces the capacity of pluripotent stem cells (CFU-S) to form surface spleen colonies in lethally irradiated mice. To exclude the possibility that such treatment interferes only with the capacity of CFU-S to form surface spleen colonies, we evaluated the capacity of CFU-S which were treated with rabbit anti-mouse brain serum (RAMBS) to restore hematopoiesis and rescue lethally irradiated mice, and to form microscopic spleen colonies. Marrow cells were treated with RAMBS or control rabbit serum. Fifty thousand treated nucleated cells were injected i.v. into lethally irradiated mice, and hematopoietic reconstitution was studied between days 8 and 15; separate groups of mice were observed for survival. We found that treatment with RAMBS impairs the capacity of marrow cells to repopulate the marrow and spleen with CFU-S, to restore blood RBC, to effect an overshoot in spleen weight, and to prolong survival; in addition it reduces the number of microscopic spleen colonies to the same extent that it reduces the number of macroscopic colonies. Hence, RAMBS appears to effect a general inactivation of CFU-S and should prove to be a useful tool in further investigations of mouse hematopoiesis.

Colony-stimulating activity in serum and bone-conditioned medium of 89Sr marrow-ablated mice.

We evaluated the levels of CSA in the serum of and in the medium conditioned by marrow-free femurs of 12 to 16-week-old female CAF1 mice whose marrows had been ablated with the bone-seeking radionuclide, 89Sr. Intact mice were studied 10 to 56 days after 89Sr injection, and mice splenectomized on days 14 and 42 after injection of 89Sr were studied on days 21 and 56, respectively. Control mice were injected with cold 89Sr; sham-splenectomized mice were used when appropriate. None of the mice had any significant levels of CSA in the serum, even the leukopenic splenectomized 89Sr-treated mice. Femur-conditioned medium from all groups contained sizable, but approximately equal, amounts of CSA; thus 89Sr marrow ablation did not adversely affect the capacity of femurs to elaborate CSA. Intraperitoneal injection of endotoxin effected an increase in serum CSA in both intact and splenectomized 89Sr marrow-ablated mice which was equal to that found in the control mice.

Rabbit anti-mouse brain serum (RAMBS): lack of specific inhibition of late committed erythroid stem cells in culture (CFU-E).

Heterologous antisera to mouse brain tissue have activity against mouse pluripotent hemopoietic stem cells (CFU-S), but not against granuloid/macrophage committed precursor cells (CFU-C). In these studies we show that anti-mouse brain serum raised in a rabbit does not possess specific activity in vitro against late committed erythroid stem cells (CFU-E).

An immunologic comparison between bone marrow and spleen-derived pluripotent hemopoietic stem cells (CFU-S) of mouse : effect of rabbit anti-mouse brain serum.

We used rabbit anti-mouse brain serum (RAMBS), with known activity against pluripotent hemopoietic stem cells (CFU-S) of mice, to compare the proportions of CFU-S from bone marrow and spleen which express the brain-associated CFU-S antigen(s). Eighty percent of marrow and 77% of spleen-derived CFU-S were inactivated by RAMBS. This suggests that these two populations of CFU-S are antigenically similar with respect to the brain-associated CFU-S antigen(s); this is in contrast to functional differences which are known to exist between the two populations of CFU-S.

Pluripotent (CFU-S) and granulocyte-committed (CFU-C) stem cells in intact and 89Sr marrow-ablated S1/S1d mice.

Peripheral blood values, femur cell counts, spleen weights, pluripotent (CFU-S) and granulocyte progenitor cell (CFU-C) concentrations and total content of spleens and femurs have been evaluated in intact (non-marrow-ablated) and 89Sr marrow-ablated S1/S1d and +/+ mice. 89Sr-irradiated mice were studied 6 and 11 days after the administration of 89Sr. In intact S1/S1d mice the femur CFU-S concentration, total femur CFU-S, femur CFU-C concentration and total femur CFU-C were 84, 54, 105 and 68% that of +/+ mice femurs respectively; the respective values for the spleens of S1/S1d mice were 40, 46, 61 and 69%. These are the first simultaneous determinations of CFU-S and CFU-C concentrations, and content of spleens and marrows, of S1/S1d and +/+ mice. In 98Sr marrow-ablated mice, 11 days after injection of the radionuclide: (a) the total content of marrow CFU-C and CFU-S was about 1% of that found in the marrows of intact mice for both +/+ and S1/S1d groups; (b) the spleens of +/+ mice increased in weight to 162% of the control, but the spleens of S1/S1d mice did not increase in weight; and (c) the spleens of +/+ mice had a total content of CFU-C and CFU-S of 800% and 260% of the control, respectively, whereas the respective values for the S1/S1d mice were 120% and 76% of the control. Thus the S1/S1d spleen fails to compensate for marrow ablation by housing additional CFU-S and has an impaired ability to compensate by housing additional CFU-C.

Hemopoiesis on macrophage-coated cellulose acetate membranes (CAMS) in mice: an immunological study.

Macrophage-coated cellulose acetate membranes (CAMS), implanted into the peritoneal cavities of sublethally irradiated mice, support the growth of hemopoietic colonies. To investigate the nature of the precursor cells (CFU-ML) which form colonies on CAMS, we pre-treated marrow cells with rabbit anti-mouse brain serum (RAMBS), a known anti-pluripotent stem cell (CFU-S) serum, plus complement (C) and studied the number of colonies formed and the distribution of their sizes among the various histological types. Marrow cells pre-treated with RAMBS + C, even with the opportunity for interaction with macrophages in vivo, did not form fewer or smaller colonies than those formed from CRS + C treated cells, suggesting that most of the CFU-ML are antigenically distinct from CFU-S.

Hematopoiesis on cellulose ester membranes (CEM). II. Enrichment of the hematopoietic microenvironment by the addition of selected cellular elements.

Cellulose ester membranes (CEM) were folded into a trilaminar open-ended tube which was implanted into the peritoneal cavity of mice. CEM rapidly acquired a stromal core with many features of marrow such as fat, fibroblasts, an abundant sinusoidal microcirculation and monocyte-macrophage-like cells. CEM took up 59iron, 99technetium sulfur colloid and produced CSF in in vitro culture but their microenvironment supported only granulopoiesis. CEM were coated on their interior surfaces with bone marrow or regenerating medullary cavity mesenchyme or bone but the stromal cores supported only granulopoiesis after 3 weeks to 3 months of implantation. CEM coated with spleen and implanted into mice developed trilineal hematopoiesis within 6 weeks with abundant erythropoiesis and megakaryocytopoiesis in addition to granulopoiesis. These CEM differed from splenic tissue in that only scattered lymphoid tissue was present. CEM coated with bone marrow and bone developed trilineal hematopoiesis but only after3--6 months of peritoneal implantation. CEM coated with regenerating medullary cavity mesenchyme failed to develop trilineal hematopoiesis. Cyclophosphamide injection did not enhance hematopoiesis. These experiments indicate that splenic, marrow and bone tissue contain stromal elements capable of being transferred onto CEM which then develop a microenvironment capable of supporting trilineal hematopoiesis.

Hematopoiesis on cellulose ester membranes (CEM). I. Functional characteristics of cells comprising the hematopoietic microenvironment.

Cellulose ester membranes (CEM) implanted into the peritoneal cavity of mice rapidly became coated with cells of peritoneal origin. Up to 56% of the cells, at a peak point 3-5 days after implantation, showed cell membrane receptors for complement and cytophilic immunoglobulin. A similar proportion of cells from CEM phagocytized yeast particles in vitro. When studied in situ, rosettes with C3b and IgG coated erythrocytes were formed by 23% of the cells coating CEM. A decreasing percentage of cells with monocyte-macrophage characteristics were detected between 5 and 17 days. CEM removed at 2 or 6 weeks after peritoneal implantation enriched tissue cultured media with colony stimulating factor which supported the growth of granulopoietic colonies in softagar culture. Mice given 59iron and 99technetium sulfur colloid i.v. showed substantial uptake of both isotopes by the CEM but the 59iron uptake could not be suppressed by hypertransfusion. Surface hematopoietic colony formation on CEM was studied 1-14 days after implantation. A peak colony number occurred at 5 days and the fell off slightly by 2 weeks. These studies indicate that the hematopoietic microenvironment of peritoneally implanted CEM contains a major sub-population of cells with monocytemacrophage features. The hematopoietic microenvironment was well-maintained even though the percentage of monocyte-macrophage marked cells decreased indicating that the microenvironment is not solely dependent upon monocyte-macrophages.

Hemopoietic support capacity of the adult mouse liver: II. Studies in acetylphenylhydrazine-treated mice.

We investigated the hemopoietic support capacity of the liver in intact and splenectomized adult mice treated with three daily injections of acetylphenyl-hydrazine (APH). Packed red cell volumes, liver and spleen weights, numbers of pluripotent hemopoietic stem cells (CFU-S) in blood and liver, and liver histology were evaluated 4,8,12,16, and 20 days after the first injection. We found that 1) splenectomized, APH-treated mice had a greater and more sustained increase in the weights of their livers than the increase found in livers of intact APH-treated mice; 2) APH treatment elicited a much greater increase in the blood and liver CFU-S of splenectomized mice (47 and 42 times normal, respectively) than it elicited in the blood and liver CFU-S of intact mice (4--5 and 4 times normal, respectively); and 3) APH treatment induced numerous foci of hemopoietic tissue in the livers of splenectomized mice. The results of the CFU-S studies can be explained by, and to some extent support, the thesis that the adult mouse liver does not support proliferation of normal CFU-S, but can trap large numbers of circulating CFU-S. In addition, these studies suggest that the livers of adult mice are able to support only limited proliferation of differentiated hemopoietic elements.

Chronic lymphocytic leukemia: correlation of clinical course and therapeutic response with in vitro testing and morphology of lymphocytes.

Forty-two patients with chronic lymphocytic leukemia (CLL) were studied for morphology of lymphocytes by light and electron microscopy (EM), in vitro responses of lymphocytes to a battery of physical and chemical agents, overall clinical status, immunologic status, course, and response to therapy. CLL lymphocytes could be classified by EM into four groups on the basis of cell size and nuclear contour and by light microscopy into two groups, small cells and large cells (lymphosarcoma cells). Patient survival did not vary with cell size or morphology as determined by light or electron microscopy. In vitro testing of CLL lymphocytes following exposure to X-ray, PHA, DMSO 2 hr at 43 degrees C, prednisolone, glutaminase, and asparaginase permitted a separation of patients into categories of normal and abnormal in vitro responses. A normal in vitro response predicted a good response to therapy but an abnormal in vitro response did not preclude a good response to therapy. Following therapy, normalization of abnormal EM morphology and in vitro response was seen in some patients. Most patients tested had decreased serum immunoglobulins and abnormal PHA responses. There was a high incidence of infections and second neoplasms. Immunologic deficits could not be correlated with variations in lymphocyte morphology or in vitro response.

Normal colony stimulating factor (CSF) production by bone marrow stromal cells and abnormal granulopoiesis with decreased CFUc in S1/S1d mice.

The concentration of CFUC and the production of stromal-derived CSF in the femora of S1/S1d mice were determined. There was a lower concentration CFUC and a smaller total number of nucleated cells in the femoral marrow of WCB6. S1/S1d (S1/S1d) mice than in WCB6. +/+/(+/+) mice or in C57B1/6. +/+ (C57Bl) mice. On the other hand stromal-derived CSF production by femora from S1/S1d mice did not differ significantly from that of +/+'s. These observations indicate that the microenvironmental defect of S1/S1d mice results in decreased growth of granulocytic precursors as well as those of erythroid and megakaryocytic cells. This is consistent with the reported decrease in multipotential stem cell proliferation. Stromal cell derived CSF production was normal and could not be implicated in the decreased production of granulocytic precursors.

Dynamics of leukemic and normal stem cells in leukemic RFM mice.

RFM mice spontaneously develop a myelogenous leukemia that is transplantable into nonleukemic RFM mice. On transplantation, hemopoietic stem cells from leukemic mice (L-CFU-S) will seed in the spleen and grow as discrete colonies, as will hemopoietic stem cells from normal mice (N-CFU-S). As the leukemic cells used in these experiments have 39 chromosomes and normal murine cells have 40, it has been possible to estimate the numbers of N-CFU-S and L-CFU-S in RFM mice at weekly intervals after these mice had been given i.v. injections of 10(6) leukemic spleen cells (spleen cells from preterminal leukemic mice). At each study time, splenic weights, peripheral blood counts, and nucleated cell counts and colony forming units (CFU-S) of marrow, spleen, and blood were assayed. The karyotypes of dividing cells from and the histology of the resultant spleen colonies were also studied. Two weeks after the injection of leukemic spleen cells, the number of CFU-S in the marrow had increased to 3 to 10 times normal, that in the spleen to 100 times normal, and that in the blood was markedly increased. Three weeks after injection, the number of CFU-S in the marrow fell from the peak level at 2 weeks, the number in the spleen rose modestly, and the number in the blood continued to be markedly increased. A normal distribution of erythroid, myeloid, and megakaryocytic colonies was obtained from CFU-S assayed 1 week after injection of leukemic spleen cells, but from CFU-S assayed 2 or 3 weeks after injection of leukemic spleen cells, the colonies formed were comprised almost exclusively of myeloid cells. From spleen colonies formed from marrow or spleen cells obtained 1 week after the injection of leukemic spleen cells, all karyotypes contained 40 chromosomes, whereas from spleen colonies formed from marrow or spleen cells obtained 2 or 3 weeks after injection of spleen cells, almost all karyotypes contained 39 chromosomes. In contrast, most of the karyotypes found in spleen colonies formed from the injection of blood cells even 3 weeks after injection of leukemic spleen cells contained 40 chromosomes. All colonies containing cells with 39 chromosomes, leukemic colonies, contained only myeloid cells. We conclude that L-CFU-S differentiate only into the myeloid series. Early in the course of the disease there is an increase in both N-CFU-S and L-CFU-S in the spleen and marrow. As the disease progresses, the numbers of N-CFU-S in both spleen and marrow decline and, during the final week of the illness, the number of L-CFU-S in the marrow declines. The CFU-S in the peripheral blood are predominantly of normal type, even late in the disease when N-CFU-S are rare in the spleen and marrow.