Marrow transfusions increase pluripotential stem cells in normal hosts.

In earlier investigations of marrow transfusions into isogeneic, nonirradiated mice, the percentage of donor cells in the recipients' marrow and peripheral blood was found to vary between 16% and 40% following transfusion of 200 million marrow cells. The present experiments demonstrated that the hosts' pluripotential stem cells were elevated to an average of 132% of simultaneously assayed nontransfused controls. The elevation persisted for two months and then returned to normal. The similar magnitude of elevation of pluripotential stem cells and of the percentage of donor cells in the recipients indicates that the seeded stem cells did not replace the hosts' own, but were added to the existing complement of pluripotential cells. Implications for the regulation of stem cell numbers are discussed.

Hematopoietic reserve provided by spleen colony-forming units (CFU-S).

Using quantitative data available from the literature on murine hematopoiesis, the functional reserve of multipotential stem cells was calculated by comparing daily blood cell production with the potential of clonogenic spleen colony-forming cells (CFU-S) to generate blood cells. The potential of the day-8 CFU-S (CFU-S-8) population is estimated to be from 4000 to 37,000 times greater than needed in steady-state hematopoiesis. The CFU-S population may thus serve to provide a functional reserve to supply large numbers of peripheral blood cells via committed lineage-specific cells within days, whenever needed.

Pluripotent stem cells with normal or reduced self renewal survive lethal irradiation.

Transfusion with 10,000 or 20,000 marrow cells resulted in 30+ days survival of 15%-50% of mice exposed to an Ld90 or LD100 or radiation. The use of congenic mice with alloenzyme markers permitted the identification of host and donor cells in the peripheral blood of transfused animals. Donor cells were present initially in all hosts. Between 55% and 92% of the animals became 100% host type by 12-24 weeks after transfusion in three separate experiments. To explore whether the temporary repopulation by donor cells was due to short-lived stem cells, the marrows of several primary hosts were transfused into secondary, lethally irradiated hosts. Some of the retransplanted primary donor and host cells persisted only temporarily. It is suggested that some of the donor stem cells in both the primary and secondary hosts had an intrinsically shortened life span.

Role of T cells in sex differences in syngeneic bone marrow transfers.

Transferred marrow cells will proliferate in normal mice not exposed to irradiation or any other type of stem cell depletion when five consecutive transfers of 40 million cells are given. Approximately 25% of the mitotic cells are of male donor origin observed cytogenetically in all of the female recipient spleens and marrow analyzed from two weeks to one and one-half years after transfusions. Male donor stem cells are accepted and form a stable component of the self-renewing stem cell pool. In contrast, only 5% female cells are found in male recipients. This sex difference in engraftment is not hormonal since castration of recipients does not alter the percentage of donor cells. Rigorous T depletion of female donor bone marrow, however, increases the percentage of donor engraftment to the level observed when male marrow, either whole or T depleted, is transferred to female recipients. The success of T-depleted female stem cells to seed male recipients is observed in both C57BL/6, a responder strain in which females readily respond to the H-Y antigen as manifest by skin graft rejection, and CBA/J, a strain in which females do not readily respond to H-Y. In addition, recipient nude BALB/c males, which lack a thymus, fail to accept whole bone marrow from BALB/c females. However, male bone marrow cells seed BALB/c nude females. These studies demonstrate that the poor engraftment of female cells in transfused male recipients is abrogated by the removal of T cells from the donor female marrow.

Hematopoietic differentiative properties of murine spleen implanted in the omenta of irradiated and nonirradiated hosts.

Whole body irradiation of the recipients of syngeneic splenic implants into the omentum greatly enchances hematopoiesis and permits survial of and repopulation by stem cells of donor origin. Donor hematopioetic stem cells do not survive in spleen implants of the nonirradiated host; irradiated hosts were therfore used in the bulk of the experiments. Differentiation in the implants of splenic fragments is predominantly erythrocytic at 10 days and shifts to predominantly granulocytic differentiation at 21 days. Suspensions of spleen cells injected into the ometntum are predominantly granulocytopioetic at 10 days. The differntiation in fragments of spleen depleted of stem cells by irradtion, seeded with bone marrow cells and implanted into the omentum results in mixed erythocytic and granulocytic hematopoiesis, with granulocytic predominance. Lymphocytic cells appeared late in the implants of irrdiated recipients even at a time of prolific lymphocytopoiesis in the host's own spleens. The cause of the delay in the implants is not clear. The data are consisent with the concept that differntiation of hematopioetic stem cells is influenced by the stromal cells of the parent organ. The erythrocytic inductive capacity of the stromal cells may be lost by mechanical disruption or modified by irraidation or a prolonged period of implantation.

Short- and long-term repopulation of lethally irradiated mice by bone marrow stem cells enriched on the basis of light scatter and Hoechst 33342 fluorescence.

Murine bone marrow subpopulations enriched in hemopoietic stem cells were transfused into lethally irradiated hosts to determine the contribution of host cells and two types of donor cells to marrow repopulation. Donor cell suspensions were a mixture of marrows from two congenic lines of mice containing electrophoretically distinguishable alloenzymes of phosphoglycerate kinase (PGK-A and PGK-B). The donor cells were sorted by high forward light scatter, low-to-intermediate perpendicular light scatter, and low Hoechst 33342 fluorescence intensity. The congenic hosts contained a third distinct marker, glucose phosphate isomerase (GPI-A). The two markers in the donor cells allowed determination of the clones generated by the seeded cells over a 36-week period of observation. The clone number declined rapidly during the first 12 weeks following transplantation and reached stable levels at 20 weeks, indicating the number of long-term repopulating cells (LTRC). The sorted subpopulation was enriched 170-fold for day-13 spleen colony-forming units (CFU-S), 235-fold for cells providing a 30-day survival, and 136- to 160-fold for LTRC. Survival for the 36-week observation period was 40%-100% for groups of hosts receiving 100-3000 sorted cells and 80% for controls receiving 2 x 10(5) unsorted cells. In all groups, similar distribution of phenotypes among peripheral blood erythrocytes, platelets, and lymphocytes at 36 weeks suggested that the repopulating donor stem cells were pluripotential. Transfusion of 3000 sorted cells, containing about 5 LTRC and 60 CFU-S, assured continuous repopulation with 95%-100% donor cells 4 to 36 weeks after transplantation, whereas significant numbers of host cells re-emerged temporarily or permanently when lower numbers of LTRC and CFU-S were transfused. The data indicate that both the quality and quantity of pluripotential stem cells in sorted bone marrow are important for complete long-term marrow reconstitution.

Mean platelet volume: the need for a reference method.

The time course of artifactual effects due to anticoagulants, specimen temperature, and interval between venipuncture and analysis on platelet volume measurements was evaluated. Split specimens were analyzed using hydrodynamic focusing, and platelet distributions were computed using a least-squares fit to a log-normal distribution. Significant artifacts resulted from exposure to EDTA, cooling to room temperature, and delay in exposure to anticoagulant. The artifactual effect of EDTA is extreme and time dependent. Collection of blood in Buffered Citrate, Acid Citrate Dextrose, or Pyridoxal-5'-phosphate supplemented Citrate yielded stable and equivalent results with rapid anticoagulation and incubation at 37 degrees C for up to six hours.

Enhanced proliferation of transfused marrow and reversal of normal growth inhibition of female marrow in male hosts 2 months after sublethal irradiation.

We have previously shown that bone marrow will seed and proliferate in normal recipients. Transfusion of 50 million cells on each of 4 or 5 consecutive days, a total of 200-250 million cells, resulted in the recipient's marrow being 20-40% of donor origin. The present paper reported on the marked enhancement of proliferation of donor cells in animals that were exposed to sublethal doses of irradiation of 300-900 R. Two months later, when their peripheral blood values had returned to normal, they were transfused with 100 million cells. The number of donor cells in the recipients exposed to 600-900 R reached 55-100% at various intervals after transfusion, with controls averaging 24% and never exceeding 40%. Since the transfused cells numbered less than 40% of the host's own complement of marrow cells, they could not replace 100% of them unless they proliferated more rapidly than the host cells. The implied competitive advantage of the donor cells was ascribed to a reduced capacity for self-renewal of the host's irradiated cells. In recipients exposed to 300 R and in nonirradiated controls, female cells failed to grow in male recipients, while male cells grew as well in female as in male hosts. The inhibition of growth of female cells in the male host was abolished by irradiation with 600 or 900 R, or by the exposure of the female donor cells to anti-Thy-1 serum and complement prior to transfusion. Experiments are under way to test the suggested immunologic nature of the inhibition phenomenon.

Reaction time performance with and without backscatter from intense pulse light.

Twenty male graduate students, 22-30 years of age, were asigned by a table of randome numbers to two groups, and visual reaction time performance with and without backscatter was measured. The subjects' task was to observe a 5 cm dial face whose needle deflected 2 mm either left or right of center. Meter deflections were either preceded by 10 light pulse from a Grimes "360 strobe or they were not preceded by light pulses. Two measures of performance were recorded: 1) voice reaction time in milliseconds, and 2) errors. Error rate (3.5%) did not discriminate between groups or conditions. Reaction time was almost twice as long with backscatter than without backscatter (1,556 ms and 854 ms, respectively). This RT increase was highly reliable statistically. Variability of RT performance increased markedly with backscatter. In practical terms, the results suggest that the effects of backscatter could induce a cumulative performace decrement in instrument scanning which might endanger air safety.