Summary:The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA CFU-GM ), pluripotent progenitors (CFUSd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA CFU-GM , CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA CFU-GM , CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA CFU-GM ) was achieved only when 10% DMSO was included with a fivestep protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.

Low O(2) concentration (1%) favors the self-renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL-6 influences both stem cells and committed progenitors at 20% O(2), we studied its effects in cultures at 1% O(2). The pre-CFC activity in Lin- population of mouse bone marrow was analyzed following 10 days of serum-free culture in medium (LC1) supplemented with IL-3 with and without IL-6, at 20 and 1% O(2) and phenotypic differentiation and proliferative history monitored. The IL-6 receptor expression and initiation of VEGF-A synthesis were also investigated. At 20% O(2), the effects of IL-6 on pre-CFC were negligible but effects on CFC were apparent; conversely, at 1% O(2), the IL-6 enhances activity of pre-CFC but not of CFC. Unlike at 20% O(2), at 1% O(2) a subpopulation of cells remained Lin- in spite of extensive proliferation. However, the absolute number of Lin- cells, did not correlate with pre-CFC activity. A relative increase in VEGF transcripts at 1% O(2) in presence of IL-3 alone was enhanced by the addition of IL-6. IL-6 enhanced pre-CFC activity at 1% O(2) and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O(2) does not exist in tissues in vivo, further studies in vitro at lower O(2) concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells.

The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-la/fl, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sublethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of ILL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-la, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/ pathological status of the organism.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

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Hamster, a hibernating animal, is an important experimental model in research on the influence of hypothermia on different physiological processes. A simple procedure for cultivation and identification of hamster hematopoetic stem cells (HSC) and hematopoetic progenitor cells (HPC) is a premise for a successful investigation upon hypothermia effects on hematopoiesis. The aim of this work was to evaluate the utilization of commercially available methylcellulose media (MC) and recombinant mouse and human cytokines for hamster HSC and HPC assays, in order to enable further studies on these cells. Hamster bone marrow mononuclear cells (BMMNC) were plated in MC containing cytokines that support mouse or human HPC growth. Also, BMMNC were resuspended in cytokine supplemented liquid media and incubated for 5 weeks with a four day monitoring of viable cell number. We demonstrated that hamster hematopoietic progenitor cells committed for erythroid lineage and myeloid lineage successfully formed recognizable colonies in both mouse and human MC, while multipotent progenitor cells formed colonies only in mouse MC. We also defined conditions for the evaluation of hamster HSC activity in liquid cultures, based on continuous 5 weeks HSC proliferation. The obtained results verify the utilization of mouse specific MC for further research on hamster HPC biology during hypothermia

Abstract. The assay system for determination of haemopoietic progenitors in peripheral blood of rats is essential for potential studies on mobilisation and transplantation of circulating progenitor cells in a rat experimental model. This paper demonstrates the possibility of detection and quantification of pluripotent progenitors (Colony Forming Units-Spleen day 8-CFU-Sd8) and committed progenitors (Colony Forming Units Granulocyte Macrophage-CFU-GM and Burst Forming Units-Erythroid-BFU-E) in peripheral blood of rats in a steady state. For determination of CFU-Sd8 the 'rat to mouse' in vivo assay was used, and for committed progenitors in vitro assays on methylcellulose were employed. The CFU-Sd8 incidence ranged from 7.3 to 11.6/ml of rat blood, similar to that reported in literature for mice. The incidence of CFU-GM was found to be 59.7 + 9.4/ml which is in the range of the literature data for mice, rabbits, dogs and humans. The incidence of BFU-E in rat peripheral blood was 4.3 _+ 1/ml, which was relatively low, but could be also considered as comparable with some literature data for dogs and humans. The CFU-E were not detected by the technique used. These results confirmed the existence of circulatory blood pluripotent progenitors (CFU-Sd8) and committed (CFU-GM and BFU-E) progenitors in rat, as has been established for some other mammalian species.

The influence of liposome structure on hematopoiesis in vivo was assessed in relation to the different contents and origins of phospholipids that make up their membrane structures. Changes within different hematopoietic cells and serum tumor necrosis factor alpha (TNF-alpha) levels were estimated up to 14 days following intravenous administration of liposomes made of either pure egg yolk phosphatidylcholine (LEY) or a soybean phospholipid preparation (LSB) into normal CBA mice. In peripheral blood, only transient changes within white blood cells were observed. In bone marrow, a persistent decline in the number of mature granulocytes, monocytes, and lymphocytes was found. The changes within femoral granulocytic proliferative compartments in various stages of differentiation and a maturation compartment pointed out that, parallel with the depletion of the granulocyte-storage pool, stimulation of de novo production of granulocytic cells occurred. Although both types of tested liposomes induced similar cellular changes, only liposomes made of pure egg yolk phosphatidylcholine induced a transient increase in serum TNF-alpha levels.