Density distribution analysis of in vivo and in vitro colony forming cells in bone marrow

The technique of buoyant density separation in gradients of Bovine Serum Albumin has been used to separate hemopoietic cell populations in mouse bone marrow that form in vivo spleen colonies and in vitro colonies of granulocytes and macrophages in an agar culture system. The density distribution profiles showed a number of reproducible density subpopulations of both in vivo and in vitro colony forming cells (C.F.C.'s). The mean density of in vitro C.F.C.'s exceeded that of the in vivo but overlap of the density profiles of the two populations was evident. Density-related differences in seeding efficiency of in vivo C.F.C.'s were observed. Freund's adjuvant treatment increased marrow and spleen in vitro C.F.C. populations. Marrow density profiles obtained three and seven days after adjuvant showed a progressive increase in in vitro C.F.C.'s in a restricted density region with no associated elevation of in vivo activity. The antimitotic agent, vinblastine, revealed differences in mitotic activity between the two cell populations, reducing the in vitro C.F.C. population to .07% and the in vivo to 5% of normal in 24 hours. Density separation of vinblastine-treated marrow produced density regions devoid of in vitro activity but containing in vivo in vivo C.F.C.'s which, upon transfer to irradiated recipients, regenerated both in vivo and in vitro density distribution profiles.     

Adherence column and buoyant density separation of bone marrow stem cells and more differentiated cells

Mouse bone marrow cells were separated by adherence column and albumin density gradient procedures, assaying for spleen colony forming units (in vivo CFU's), agar colony forming cells (in vitro CFC's) and cluster forming cells. Column filtrates were enriched for CFU's whereas in vitro CFC's and cluster-forming cells were enriched in adherent fractions. Gradient separation of these column fractions gave density distribution profiles indicating the non-identity and heterogeneity of CFU's and in vitro CFC's.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

Purification and characterisation of the in vitro colony forming cell in monkey hemopoietic tissue

Buoyant density gradient separation of Rhesus monkey bone marrow, spleen and blood leukocytes has demonstrated a reproducible and homogeneous light density distribution profile of cells capable of forming hemopoietic colonies in agar culture (in vitro colony forming cells — CFC). High resolution density gradient separation performed on a light density fraction of bone marrow produced on average a 100-fold enrichment of in vitro CFC with the most enriched fractions containing the majority of the in vitro CFC population present in the original marrow. Fractions were routinely obtained in which up to 23% of cells formed colonies and 33% were capable of proliferating to some degree upon stimulation. Tritiated thymidine suiciding showed the active proliferative status of the in vitro CFC and application of autoradiography and morphological characterisation to highly enriched density fractions has shown that the in vitro CFC in normal marrow is a transitional lymphocyte. Single cell transfer experiments have shown that in vitro CFC's formed colonies containing both granulocytes and macrophages, formally demonstrating the clonal origin of in vitro colonies and the common origin of granulocytes and macrophages.     

ERYTHROID AND GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN PRE-NATAL 'STEEL' (SlJ/SlJ) ANAEMIC MICE

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Sl^J/Sl^J mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Sl^J/Sl^J foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when ‘normal’ livers contain approximately 6 × 10⁵ erythroid colony forming cells/liver. In Sl^J/Sl^J fetuses the maximum reached is only 1 × 10⁵. Granulocyte-macrophage colony forming cells (CFU_C) in Sl^J/Sl^J fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Sl^J/Sl^J mice in the 2–3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Sl^J/Sl^J mice, but their production in the Sl^J/Sl^J pre-natal spleen appears unaffected.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

In vitro and in vivo activity of 4-thio-uridylate against JY cells, a model for human acute lymphoid leukemia

We have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s⁴UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s⁴UMP on JY cells. Treatment of JY cells with s⁴UMP suppressed their colony forming activity and induced apoptosis; healthy human bone marrow granulocyte–macrophage progenitor cells were 14-fold less sensitive to the nucleotide. In vivo effectiveness of s⁴UMP was determined using xenograft SCID mouse model. s⁴UMP decreased the cell number and colony forming activity of the total cell content of the femur of SCID mice transplanted with JY cells without affecting the bone marrow of healthy mice. These results suggest that s⁴UMP alone or in combination with other clinically approved anti-leukemic remedies should be further explored as a potential novel therapeutic agent.     

Comparative Investigation of Mesenchymal Stem Cells Isolated from the Bone Marrow and Fetal Liver of Mouse and Rat

We studied the properties of cells forming fibroblast colonies from the bone marrow and fetal liver of mouse and rat. Bone marrow and fetal liver cells formed colonies in vitro including fibroblasts as well as a considerable proportion of macrophages. The colonies formed from bone marrow and hepatic cells of rat differed from the murine ones by a higher proportion of fibroblasts. Most colonies derived from the bone marrow of both mouse and rat included a fraction of cells expressing alkaline phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the fetal liver included low proportions of such cells. The cell layers derived from the colony-forming fibroblasts of both bone marrow and fetal liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicated that these progenitor cells can form hematopoietic microenvironment.     

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

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

ANALYSIS OF PROLIFERATION AND DIFFERENTIATION OF FOETAL GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN HAEMOPOIETIC TISSUE*

Analysis of in vitro colony formation in agar cultures of foetal haemopoietic tissues of eight mammalian species has shown that granulocyte-macrophage progenitor cells are present in foetal liver, yolk sac, marrow and spleen in numbers approaching the incidence in adult marrow. Such characteristics as buoyant density, growth rate and differentiation served to distinguish foetal from adult colony forming cells (CFCs). Cell cycle analysis performed by exposing haemopoietic cells to high doses of tritiated thymidine in vitro showed that foetal CFC proliferation in species of short gestation (rabbit, rat, mouse) approached or exceeded that observed in adult marrow. In contrast, in species of long gestation (human, monkey, calf, lamb, guinea-pig) a period of variable duration was observed when foetal liver CFCs entered a non-cycling G₀ or blocked G₁ phase. In these species foetal liver CFCs were found to be proliferating actively early in gestation and following the non-cycling phase again re-entered a proliferative state associated with onset of active granulopoiesis in foetal marrow and possible migration of CFC from liver to marrow. These results indicate the existence of granulocyte-macrophage progenitor populations displaying foetal characteristics and adapted to particular stages of haemopoietic development, a situation which closely parallels that reported for erythropoiesis.     

THE EFFECT OF CYTOSINE ARABINOSIDE IN VITRO ON AGAR COLONY FORMING CELLS AND SPLEEN COLONY FORMING CELLS OF C57BL MOUSE BONE MARROW

This study reports the effect of cytosine arabinoside in culture on two classes of bone marrow progenitor cells in C57BL mice, agar colony forming cells (ACU) and spleen colony forming cells (CFU). Both normal cells and rapidly proliferating cells were studied. The results show that in normal mice, 23 % of ACU but only 7 % of CFU are killed following 1 hr incubation with the drug. With longer periods of incubation, the survival of ACU in the controls is poor, and the results for the drug-treated cultures suggest that the cells are held up in cycle. In continuously irradiated mice, the proportion of ACU and CFU killed after 1 hr incubation with drug is increased to 43–54%, confirming previous results that these cells are proliferating more rapidly than in normal mice. In mice treated with myerlan, 54 % of ACU are killed by 1 hr in vitro exposure to cytosine arabinoside, again confirming that ACU are rapidly proliferating. However, the proportion of CFU killed is lower (23 %). These results are compared with other studies of the effect of cytosine arabinoside in vivo and also with thymidine suicide in the same strain of mice. The results show that cytosine arabinoside has the same effect as tritiated thymidine, and also that the proportion of CFU killed by these agents in vitro is lower than when the agents are injected in vivo. It is suggested that the conditions in culture have an adverse effect on CFU, which cease DNA synthesis, and are protected from the killing effect of cytosine arabinoside and tritiated thymidine. Since cytosine arabinoside in vitro has an effect similar to tritiated thymidine in vitro on bone marrow progenitor cells in C57BL mice, in vitro incubation with cytosine arabinoside could be an alternative method to thymidine suicide for measuring differences in cell proliferation rate.     

Sedimentation velocity characterisation of the cell cycle of granulocytic progenitor cells in monkey hemopoietic tissue

Sedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H³TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H³TdR exposure indicates that 55% of total CFC's in adult marrow are in G₀ or G₁ with a volume of 410 μ³, 42% are in S phase and of volume 450–950 μ³, and the remainder are in G₂ and mitosis with a volume of between 600–950 μ³. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G₂ component. H³TdR exposure confirmed the non-cycling status of these fetal progenitor cells.     

FETAL HEMOPOIESIS IN DIFFUSION CHAMBER CULTURES

The growth pattern of fetal liver (FL), normal adult bone marrow (NABM) and regenerating (post Velban treatment) adult bone marrow (RABM) colony forming units (CFU) cultured in diffusion chambers (DC) was studied. When twenty CFU were implanted into DC the recovery of CFU after 4 days with FL, NABM or RABM was 133 ± 7, 19 + 2 and 34 ± 2 CFU, respectively. The transplantation fraction of CFU from NABM decreased from 10-4% on day 0 to 6–9 % on day 4; that of FL did not change from the initial 6-2%. The growth rate of CFU derived from FL was substantially greater than that from NABM. The relative growth of FL and RABM CFU was clearly inhibited when the concentration of cells cultured was increased. Spleen colonies from FL cells before culture were larger (P < 0–005) than colonies from NABM but after 7 days of culture there was no difference between the two groups. Histological examination of spleen colonies showed that after DC culture FL and NABM CFU were differentiating along the three normal pathways. These data suggest that intrinsic differences exist between fetal and adult stem cells in the in vivo diffusion chamber culture system.     

Cell proliferation in Xenopus tissues: A comparison of mitotic incidence in vivo and in organ culture

Using the Colcemid technique, the mitotic incidence (MI) was measured in the epidermis, lung, spleen, liver, kidney and ovarian follicular cells of metamorphosed, immature Xenopus laevis laevis. The MI was higher at 25°C than at 20°C, and there was a significant ranking correlation between organs in respect of the MI in different animals. With the exception of the liver and kidney, organ cultures showed good preservation for up to six days in vitro using a medium supplemented with 10% fetal calf serum, and values for MI comparable with or even higher than in vivo were obtained.     

SERUM LEVELS OF COLONY STIMULATING FACTOR(S) AND THE GROWTH OF BONE MARROW CELLS IN DIFFUSION CHAMBERS

Fluctuations in the body fluids of long-ranged humoral substance(s) capable of stimulating the growth of bone marrow granulocytic and macrophage-like cells in diffusion chamber cultures in vivo, was observed after whole body irradiation of mice. The fluctuation pattern was similar to that of the in vitro colony stimulating factor(s) of the sera of irradiated mice which indicates a relation between in vivo and in vitro active factor(s).     

The in vitro differentiation of density sub-populations of colony-forming cells under the influence of different types of colony-stimulating factor.

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density subpopulations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post-endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macrophage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

Expression of congenital defects in the haemopoietic micro-environment. Erythroid and granulocyte-macrophage progenitor cells in pre-natal 'steel' (Slj/Slj) anaemic mice.

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Slj/Slj mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Slj/Slj foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when 'normal' livers contain approximately 6 X 10(5) erythroid colony forming cells/liver. In Slj/Slj fetuses the maximum reached is only 1 X 10(5). Granulocyte-macrophage colony forming cells (CFUc) in Slj/Slj fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Slj/Slj mice in the 2-3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Slj/Slj mice, but their production in the Slj/Slj pre-natal spleen appears unaffected.     

Mast cell growth factor (C-Kit Ligand) in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3:in vivo hemopoietic effects in irradiated mice compared toin vitro effects

In the presence of hemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has proven to be a potent hemopoietic regulatorin vitro. In these studies, we examined thein vivo effects of MGF in combination with GM-CSF or GM-CSF plus IL-3. Effects were based on the ability of these cytokines to stimulate recovery from radiation-induced hemopoietic aplasia. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of⁶⁰Co radiation followed by subcutaneous administration of either saline, recombinant murine (rm) MGF (100g/kg/day), rmGM-CSF (100g/kg/day), rmIL-3 (100g/kg/day), or combinations of these cytokines on days 1–17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-s), granulocyte macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC) and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF administered in combination with GM-CSF or in combination with GM-CSF plus IL-3 either produced no greater response than GM-CSF alone or down-regulated the GM-CSF-induced recovery. These results sharply contrasted results ofin vitro studies evaluating the effects of these cytokines on induction of GM-CFC colony formation from bone marrow cells obtained from normal or irradiated B6D2F1 mice, in which MGF synergized with GM-CSF or GM-CSF plus IL-3 to increase both GM-CFC colony numbers and colony size. These studies demonstrate a dichotomy between MGF-induced effectsin vivo andin vitro and emphasize that caution should be taken in attempting to predict cytokine interactionsin vivo in hemopoietically injured animals based onin vitro cytokine effects.Abbreviations GM-CSF Granulocyte-Macrophage Colonly-Stimulating Factor - IL-3 Interleukin-3 - MGF Mast Cell Growth Factor - SCF Stem Cell Factor - rm Recombinant Murine - CFU-s Colony Forming Unit-Spleen - GM-CFC Granulocyte Macrophage Colony-Forming Cell - WBC White Blood Cells - RBC Red Blood Cells - PLT Platelets - SLF Steel Factor - G-CSF Granulocyte Colonly-Stimulating Factor - IL-1 Interleukin-1 - IL-6 Interleukin-6 - Epo Erythropoietin - CFC Colony-Forming Cell - Sl Steel - BFU-e Erythroid Burst Forming Units - s.c Subcutaneous - PEG Polyethyleneglycol - PIXY321 GM-CSF/IL-3 Fusion Protein