I. Fehér

Blast Colony Forming Cell-Binding Capacity of Bone Marrow Stroma from Myelodysplastic Patients

A specific stroma function can be quantitatively assessed by counting the stroma‐adherent blast cell colonies (CFU‐BL) that are formed from normal plastic nonadherent mononuclear bone marrow cells (PNAMNC) after a short‐term coincubation (“panning”) with the preformed stromal layer. In order to obtain information of stroma function in myelodysplasia (MDS), the “CFU‐BL‐binding capacity” of stroma from normal bone marrow and from patients with MDS were compared. Stromal cell cultures were established from mononuclear bone marrow cells in microplate cultures cultured with or without 10−6 M hydrocortisone. CFU‐BL‐binding capacity was studied by counting blast colonies seven days after panning, and the results were expressed as CFU‐BL/103 PNAMNC. Normal marrow stromal layers bound CFU‐BL only if they were cultured with hydrocortisone, while MDS stromal layers also bound CFU‐BL in the absence of hydrocortisone. For further studies of the function of MDS stroma, the effect of growth factors (stem cell factor [SCF], G‐CSF, interleukin 3 [IL‐3] and their combinations) on CFU‐BL binding by normal or MDS stroma has also been compared. Twenty‐hour incubation of the stromal layers with a standard dose (100 ng/ml) of various hemopoietic growth factors (IL‐3 alone or in combination with SCF, G‐CSF alone or in combination with SCF) did not have any effect on CFU‐BL binding by normal marrow stroma, but increased the CFU‐BL binding by stromal layers from MDS bone marrow. These findings suggest that although stromal microenvironment in MDS is capable of supporting hemopoiesis, bone marrow stroma from MDS patients differs in some characteristics from the normal stroma.

Proliferation Inhibition of Murine Pluripotent Haemopoietic Stem Cells By Interferon Or Poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3·36 Gy (336 rad) 60Co‐gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10–11 M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C‐treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non‐cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.

Differences in the growth and differentiation of the CFU fraction surviving sublethal irradiation.

The following characteristic changes of self-reproduction, growth and differentiation of CFU surviving 300 R60 Co irradiation in vivo were found: (1) CFU duplication from IBM started 24 hr earlier than from CBM; (2) Colonies from ICFU showed a more rapid growth at the beginning than colonies from control CFU; and (3) The ratio of erythroid: granuloid (E:G) colonies decreased and more megakaryocytic colonies were found in the IBM-transplanted group. The change in the E:G ratio proved to be transient. The more rapid growth of colonies and the earlier start of CFU duplication may be explained by the heterogeneity of the pluripotential CFU population. The possible selection of a more radioresistant CFU fraction is suggested.

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: A murine model for human bone marrow purging

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.

Correlation between circulating stem cell count and stem cell regeneration in locally irradiated bone marrow.

Stem cell regeneration in 650 R locally irradiated mouse bone marrow was studied as a function of the blood CFU level. The number of circulating stem cells was kept at a low level by total body irradiation (the locally irradiated tibia excepted) or at a high level by repeated endotoxin injections. A certain correlation between the number of circulating CFU and regeneration of CFU in locally irradiated bone marrow has been found. In the locally irradiated tibias of otherwise nontreated mice, with slightly elevated blood CFU level, CFU content was 38% of the normal value on day 12 after irradiation. In the experimental groups with a blood CFU level lower than the normal, 5-17% CFU regeneration was found. In mice with a circulating CFU number two to three times higher than the normal, CFU regeneration reached 53% of the normal level. A possible role of circulating CFU in the regeneration of locally irradiated, partially damaged bone marrow is suggested.

PROTECTION OF CYCLING CFUs AGAINST HYDROXYUREA BY LOW DOSES OF ACTINOMYCIN D

Low dose (80 μg/kg) Actinomycin D (AD) produced a significant but transient inhibition of proliferation of the haemopoietic stem cells (CFUs) in chimaeras or in mice regenerating after sublethal irradiation. The same dose of AD had no effect on the resting CFUs population. During the period of proliferation inhibition, CFUs proved to be insensitive to the killing effect of [3H]thymidine in vitro and hydroxyurea (HU) in vivo. In Ehrlich ascites tumour (EAT) bearing mice enhanced CFUs turnover rate was found. Eighty μg/kg AD produced a selective effect in these mice: it protected the proliferating CFUs population without diminishing the effect of hydroxyurea on the tumour cells.

Stromal cell growth and blast colony-forming cells in AML bone marrow

Bone marrow cells (BMC) from normal donors and from patients with acute myeloid leukaemia (AML) were cultured. Growth kinetics and the efficiency of stromal layers in supporting the adhesion of normal blast-colony forming cells (BL-CFCs) were studied. BMC from treated AML patients formed confluent stromal layers faster than normal BMCs. BL-CFC binding capacity of normal and AML stromal layers did not differ: on normal stromal layers 67.3-147, on AML stromal layers 63-117 colonies per 5 x 10(5) plastic non-adherent BMC were formed. The amount and/or binding capacity of BL-CFCs was found to be normal in two AML patients in complete remission, while a significantly reduced number and/or binding capacity of BL-CFCs was found in AML non-treated patients and in patients within 4 weeks after the last cytostatic course.

Growth kinetics and blast-colony forming cell binding capacity of aplastic anaemic stromal cells

The kinetics of bone marrow cell growth and a special function of stromal cells (the capability of binding blast colony forming cells) were studied in patients with aplastic anaemia (AA). All 10 patients studied showed faster growth of bone marrow stromal cells. The time for a confluent stromal layer formation was 24.5 days for AA bone marrow as opposed to 33.0 days for normal bone marrow. This faster growth rate could also be observed if normal bone marrow cells, depleted of plastic non-adherent fraction, were plated, suggesting that at least one of the reasons for altered stromal cell growth kinetics in AA is the changes in the ratio of plastic adherent/non-adherent cells. Functionally, i.e. in supporting the growth of normal bone marrow blast colonies, AA stromal layers did not differ from that of normal stromal layers, independently of the clinical state of the disease (AA or SAA; in one patient before or after ATG treatment; in two patients after successful allogenic bone marrow transplantation). Moreover, in some AA patients this blast colony forming cell binding function of AA stromal layers could also be detected in samples cultured without hydrocortisone (i.e. in the absence of fat cells), suggesting that AA stroma also differs qualitatively from normal stroma without inducing a defective microenvironment for stem cell homing.

Study of the Radioprotective Effect of 5-methoxytryptamine on Haemopoietic Stem Cells

SummaryThe radioprotective effect of 5-methoxytryptamine (Mexamin) was studied on the survival of colony-forming cells. The exogenous method was used in the dose-range 195 to 389 rads, and the endogenous method in the range 486 to 925 rads. The combination of the two methods makes possible reliable determination of the radioprotective effect of Mexamin both in the low and high dose-range. The slope of the curves suggests that the radioprotective effect of Mexamin is not a simple dose-reduction effect.