PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.     

THE EFFECT OF E TYPE PROSTAGLANDINS ON THE PROLIFERATION OF HAEMOPOIETIC STEM CELLS IN VIVO

The ability of protaglandins E₁ and E₂ to stimulate the proliferation of haemopoietic stem cells (CFU_s) was studied in vivo. PGE₂, in a dose range of 10^-4 to 10^-1μg/g body weight and PGE₁ in a dose range of 10^-5 to 10^-1μg/g body weight, produced a rapid cycling wave of CFU_s. The increase in the number of CFU_s in S phase was not followed by a rise in the femoral CFU_s content, and except for a transient increase in femoral CFU_c level, no increase in differentiation was found either. Therefore, it is proposed that haemopoiesis after PG-induced CFU stimulation is ineffective. PGE₂ did not stimulate regeneration of CFU_s in a perturbed state (after sublethal irradiation). All these findings support the idea that PGEs might represent potent stimulators of the haemopoietic stem cells acting in physiological doses. However, if acting concurrently with physiological control systems PGs lead to ineffective haemopoiesis (under normal conditions) or do not exert any measurable effect (after sublethal irradiation).     

THE EFFECT OF NITROGEN MUSTARD ON THE HAEMOPOIETIC STEM CELLS OF THE BONE MARROW IN THE RAT

The sensitivity of haemopoietic stem cells to the action of nitrogen mustard has been investigated by transfusion of bone marrow from treated donor rats to recipients whose own haemopoiesis had been reduced to low levels by whole body X-irradiation. By measurement of the resultant erythropoiesis in the recipients with radioactive iron, a comparison of the repopulating ability of nitrogen mustard treated bone marrow with that of normal bone marrow could be made. It was found that although a dose of 0.9 mg/kg body weight reduces bone marrow cellularity to less than 10% of normal, repopulating ability is not decreased to much less than half the normal level. This is in contrast to the effects of X-radiation, which has a more marked effect on the stem cell population than on the differentiated marrow cells. Possible reasons for this difference are discussed. It could be that the proliferative or metabolic state of the cell plays a role, or that some repair mechanism is operative in the stem cells which does not exist in the differentiated cells.     

AGE DEPENDENCE OF THE NUMBER OF THE STEM CELLS IN HAEMOPOIETIC TISSUES OF RATS

The number and concentration of haemopoietic stem cells in the femoral bone marrow and spleen of Wistar rats of different ages were investigated. Stem cells were assayed by the spleen colony technique in irradiated rat recipients. The ability of the recipient spleen to harvest transplanted tissue as a macroscopic colony was found to be dependent on the recipient's age. Changes with senescence were observed also in the concentration and the size of the stem cell compartment both in the marrow and spleen. No differences were demonstrated in the seeding of transplanted colony-forming units into the spleen of recipients of 1 and 4 months of age. A rats-mice strain difference in the effect of senescence on the haemopoietic stem cells is discussed.     

KINETIC PROPERTIES OF HAEMOPOIETIC STEM CELLS

A comparison of the exocolonizing and autorepopulating tests for haemopoietic stem-cell assay indicate that the ‘overshoot’in splenic colony formation, observed 12–14 days after 150 rad total-body radiation (TBR), only occurs with the auto-repopulation assay. The explanation is that the priming dose of 150 rad increases the absolute seeding rate of stem cells from the marrow. A seeding rate significantly greater than normal can ‘take’only if the spleen is available—it can expand and accommodate stem cells while the bone marrow cannot. If, however, the absolute number of colony-forming cells are decreased in the femur, a relative increase in seeding rate can take place even in the splenectomized animal. Evidence is presented concerning the different turnover states of exo- and autorepopulating stem cells (CFU) and those responsible for erythropoietic response (ERC), and the precursors of agar colony-formers.     

STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

ORGANIZATION OF HAEMOPOIETIC STEM CELLS: THE GENERATION-AGE HYPOTHESIS

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 12 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half, to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

HAEMOPOIETIC STEM CELLS IN EXPERIMENTAL HAEMOLYTIC ANAEMIA

Phenylhydrazine treatment produced an expansion of spleen CFU (10 x N) and an even greater expansion of spleen ACFU (40 x N). Blood CFU and ACFU levels also increased. While marrow CFU did not change marrow ACFU showed a pronounced drop. The ratio of ACFU/CFU varied between tissues, it was highest in marrow and lowest in peripheral blood. The changes in CFU, ACFU and blood granulocytes are discussed in relation to the radioprotective action of phenylhydrazine pretreatment of mice.     

REGULATION OF HAEMOPOIETIC STEM CELL TURNOVER IN PARTIALLY IRRADIATED MICE

The existence of a possible local control of CFU turnover was studied in mice in which one tibia only was irradiated (LR mice) and in mice in which one tibia was shielded during whole-body irradiation ('TBR'mice). In both the LR and 'TBR'mice, the increased CFU turnover continued in the irradiated tibiae even after the time when in the unirradiated (shielded) tibiae it returned to normal levels. The early temporary decrease in the CFU numbers in the shielded tibiae of 'TBR'mice is attributed to a temporary demand for increased differentiation rather than to migration of CFU. The direct control of CFU turnover appears to be local in nature, in contrast to the stimulus for CFU differentiation.     

THE INFLUENCE OF DIETARY PROTEIN DEFICIENCY ON HAEMOPOIETIC CELLS IN THE MOUSE

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1 -4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs–although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18 %) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstitutive role during the refeeding process.     

THE ENHANCEMENT OF HAEMOPOIETIC STEM CELL RECOVERY IN IRRADIATED MICE BY PRIOR TREATMENT WITH CYCLOPHOSPHAMIDE

Studies are reported of the enhancement of stem cell recovery following whole body irradiation as a result of prior administration of cyclophosphamide. It is shown that the much larger enhancement of regeneration observed for the hosts own surviving stem cells, compared to the regeneration of injected bone marrow stem cells, is due to the different numbers of stem cells initiating the regeneration in conjunction with the time course of stem cell regeneration. The results show that the environmental changes produced by cyclophosphamide greatly enhance haemopoietic recovery even though at the dose used this agent is relatively toxic to stem cells. Furthermore it has been shown that the level of stem cell regeneration is nearly independent of the γ-ray dose in the range 3–8 gray (300–800 rad). If human bone marrow should respond similarly it follows that regeneration produced by cytotoxic drugs administered prior to radiation embodies a considerable safety factor as far as recovery of the haemopoietic system is concerned.     

天花粉对小鼠子宫肥大细胞超微结构的影响

本研究用雌性中国１号小鼠１２只,分实验组和对照组,实验组分别腹腔注射天花粉后处死,取子宫组织作超薄切片,电镜观察。在天花粉作用下,常见子宫肥大细胞脱颗粒,呈功能活化状态,并与子宫平滑肌细胞紧密相邻。提示肥大细胞可能通过脱颗粒释放组胺促进子宫平滑肌的收缩。同时,也见肥大细胞与子宫结缔组织中的成纤维细胞,淋巴细胞等密切接触,提示成纤维细胞和淋巴细胞与天花粉作用下子宫肥大细胞的数量增多有关。     

小鼠造血干细胞增殖与分化性能的研究

本文选择Ｙ染色体特异的性别决定基因作为新的细胞遗传标志,采用ＰＣＲ技术研究了小鼠造血干细胞的增殖与分化性能。将雄鼠骨髓细胞输注给经致死剂量射线照射的雌性受体小鼠、ＰＣＲ测试结果表明,所有ＣＦＵ－Ｓ均为供体起源。供体来源的ＣＦＵ－Ｓ在其输入体内后,可通过增殖,分化形成各系造血细胞,但ＣＦＵ－Ｓ中的纤维母细胞和ＣＦＵ－Ｓ重建造血后受体小鼠骨髓中的纤维母细胞均为受体起源。由此可见,小鼠骨髓中的ＣＦＣ－Ｓ     

SOME COMPARISONS OF THE KINETIC PROPERTIES OF FEMORAL AND SPLENIC HAEMOPOIETIC STEM CELLS

A slightly different radiosensitivity is confirmed between colony forming units (CFU) in the femur as opposed to those of the spleen. The recovery pattern of the CFU in the femur and in the spleen following sublethal doses of radiation can be markedly different, depending on the radiation dose, particularly concerning the postirradiation ‘dip’. The turnover state of splenic CFU appears to be greater in normal animals than that of the femoral CFU. Hypertransfusion doubles the splenic CFU by the fourth day, while only marginally affecting femoral CFU. Erythropoietin, while increasing the CFU turnover significantly in both femur and spleen of the polycythaemic animal, also increases femoral but not splenic CFU.     

低分子量硫酸葡聚糖对小鼠造血干细胞动员作用的研究

给小鼠静脉注射低分子量（＜１０￣４ｕ）硫酸葡聚糖（ＤＳ）１５ｍｇ／ｋｇ后外周血中白细胞、单个核细胞（ｍｏｎｏｎｕｃｌｅａｒｃｅｋ,ＭＮＣ）、ＣＦＵ－ＧＭ、ＢＦＵ－Ｅ和ＣＦＵ－Ｍｉｘ产率等指标出现时相性变化。给药后１ｈ开始升高,２ｈ达到高峰、分别为药前值的２．２、２．６、３．８、４．４和３．０倍,７ｈ时趋向正常。给药后２ｈ上述各类细胞在外周血中的含量随着ＤＳ的剂量增加而增加。白细胞、ＭＮＣ计数在ＤＳ１８０ｍｇ／ｋｇ时达到峰值,均为对照组的４倍。２４０ｍｇ／ｋ８时未见明显增加。不同剂量ＤＳ对各系祖细胞均有不同程度的动员作用,ＤＳ剂量１５－３０ｍｇ／ｋｇ效果最好,每升血中ＣＦＵ－ＧＭ、ＢＦＵ－Ｅ、ＣＦＵ－Ｍｉｘ的数量分别相当于对照组的５．０、１１．９和８．８倍。其峰值出现时间与白细胞、ＭＮＣ不同,表明ＤＳ对不同类型细胞的作用机制也不尽一致。经口给小鼠投以ＤＳ２４０和４８ｏｍｇ／ｋｇ后,未见外周血中白细胞、ＭＮＣ计数有显著性升高,提示对造血干细胞没有动员作用。     

A DEFICIENCY OF HEMATOPOIETIC STEM CELLS IN STEEL MICE

In the present study, population sizes of high self-renewal potential stem cells, i.e. colony forming units (CFU), and low self-renewal potential stem cells, i.e. transient endogenous colony forming units (TE-CFU) in Sl/Sl^d mice and their normal congenic littermates were measured and compared. By correcting for differences in the seeding efficiency ‘f’, it was possible to demonstrate that Sl/Sl^d mice suffer a deficiency of both stem cell populations. Therefore, it is concluded that the defective stromal tissue of the Sl/Sl^d mouse does not support normal size stem cell populations. However, as noted in the discussion, it remains an open question as to whether the defective stromal tissue supports normal erythroid differentiation at the stem cell level.     

EFFECTS OF HUMAN LEUKOCYTE CONDITIONED MEDIUM ON MOUSE HAEMOPOIETIC PROGENITOR CELLS

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells. but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s. an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

MOBILIZATION OF HAEMOPOIETIC STEM CELLS (CFU) INTO THE PERIPHERAL BLOOD OF THE MOUSE; EFFECTS OF ENDOTOXIN AND OTHER COMPOUNDS

Factors affecting the circulation of haemopoietic stem cells (CFU) in the peripheral blood of mice were investigated. I.v. injection of sublethal doses of endotoxin, trypsin and proteinase appeared to raise the number of CFU per ml blood from about 30–40 to about 300–400 or more within 10 min. The effect was smaller when smaller doses of the substances were injected. After this initial rise the number of circulating cells returned to normal in a few hours. Following endotoxin there was a second rise which started 2–3 days after injection and attained a peak on the 6th–7th day. The first rise is explained as a mobilization of stem cells from their normal microenvironments into the blood stream; the second rise is considered to reflect proliferation of CFUs in the haemopoietic tissues. The spleen seems to be acting as an organ capturing CFUs from the blood and not as a source adding stem cells to the blood.
The early mobilization of CFU after endotoxin injection did not coincide with a mobilization of neutrophils. The number of circulating band cells was increased during the first hours.
The importance of 'open sites'in the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal X-irradiation and injecting normal bone marrow cells. When a greater number of syngeneic bone marrow cells was injected intravenously, the level of circulating CFU in irradiated mice was slightly lower than the level in unirradiated mice during the first hours.