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.     

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).     

Survival of human bone marrow progenitor cells after freezing: Improved detection in the colony-formation assay

If cryopreserved suspensions of human bone marrow were stimulated by human placental conditioned medium in the same way as fresh unseparated marrows, less than 40% of granulopoietic progenitor cells (CFU_c) was identified. By adding α-thioglycerol (0.6 mM) to the culture medium, the concentration of detectable CFU_c in cryopreserved bone marrow was increased by a factor of 3.4, and the recovery of CFU_c after cryopreservation rose to 90%. The low recovery of CFU_c after freezing in the absence of α-thioglycerol is due to the destruction of accompanying cells. Noncolony-forming cells normally present in the fresh human marrow promote colony formation in cultures stimulated by placental conditioned medium. Their effect can be replaced by α-thioglycerol. It is concluded that, in order to detect all CFU_c independent of the cellular composition of the marrow suspension, this supplement is essential for CFU_c cultures stimulated by conditioned medium.     

Stimulation of differentiation and proliferation of haemopoietic cells in vitro

A liquid culture system, for haemopoietic cells, has been developed using bone marrow cells alone, or co-cultures of thymus and bone marrow cells, inoculated into four ounce medical bottles. After several days growth, such cultures consisted of an attaching population of cells, forming discrete colonies, and a non-attaching population. In the (co-cultures) there was a 2 X enhancement of monolayer colony development compared with the combined total present in the (marrow alone) plus (thymus alone) cultures. Also, better maintenance of non-attaching cells was seen in the (co-cultures). Normal CFUS and CFUC were present in both the (marrow alone) and the (co-cultures) for at least 14 days. In the (marrow alone) cultures, granulocytes in all stages of development were present for the first week, but by 12 days the culture consisted mainly of mono-nuclear cells. In the (co-cultures), however, at 12 days more than 60% of the cells were granulocytes, in all stages of differentiation. (Co-cultures) established using lethally irradiated thymus cells were not able to support this prolonged myeloid differentiation. By feeding the (co-cultures) it was possible to maintain production of (granulocytic) cells for at least ten weeks, although no fully mature granulocytes were observed. After the second feeding, no CFU_S were detectable, but variable numbers of agar colony forming cells (not classical CFU_C) were present at least for ten weeks.     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

MEASUREMENT OF THE DURATION OF DNA SYNTHESIS IN ERYTHROID CELLS OF THE BONE MARROW USING A DOUBLE LABELLING AUTORADIOGRAPHIC TECHNIQUE DESIGNED SPECIFICALLY FOR USE WITH HAEMOPOIETIC TISSUES

This paper describes a double labelling autoradiographic technique for use with haemopoietic tissues. In involves two photographic emulsions separated by a thin piece of mica on which the cells have been smeared. In this way the autoradiographic grains due to tritium and carbon-14 appear above and below the cells respectively. Applying the method to bone marrow normoblasts of young rats, the average duration of DNA synthesis (t_s) for the pro- and early normoblasts taken together is found to be 5.1 hr and the mean cell cycle time (t_c) to be 8.2 hr. For the intermediate normoblasts, the corresponding figures are 6.3 hr and 15.7 hr. Average values for all dividing normoblasts in the bone marrow are 5.8 hr and 12.8 hr respectively for t_s and t_c. The average duration of mitosis is 32 min.     

Bone Marrow Response to Damage Induced By Hydroxyurea Or Colchicine

The extent of bone marrow damage caused by the administration of single or repeated doses of either hydroxyurea (1000 mg/kg b.w.) or colchicine (1 mg/kg b.w.) are comparable. This conclusion is based on serial studies of bone marrow cellularity and of the CFUc numbers in the bone marrow. the proliferation response of the pluripotential haemopoietic stem cells, determined by the cells forming colonies in the spleen of lethally irradiated mice (CFUs) markedly differs if the bone marrow damage is caused by hydroxyurea or colchicine. While hydroxyurea administration stimulates a large proportion of the resting G₀ cells into the cell cycle, the damage induced by colchicine is followed by only a mild increase in the CFUs proliferation rate. The seeding efficiency of the spleen colony technique has been determined after both hydroxyurea and colchicine administration. This parameter, important for the estimation of the number of the pluripotential haemopoietic stem cells in blood forming organs, is significantly affected by hydroxyurea administration, but also by repeated injections of colchicine. Following a single dose of hydroxyurea, the time-course of the CFUs numbers, which were corrected for the change in the seeding efficiency, shows an overshoot occurring after 18–20 hr. At the other time periods, the number of pluripotential haemopoietic stem cells is little affected by a single hydroxyurea injection. This poses a question about the nature of the stimulus, which after hydroxyurea administration triggers the CFUs from the resting G₀ state into the cell cycle. There is evidence that this stimulus is probably not represented by the damage caused to the various intensively proliferating cell populations of the bone marrow. This evidence is based on experiments which show that colchicine induced damage, of a degree similar to that after hydroxyurea, does not stimulate the CFUs proliferation rate to an extent comparable to hydroxyurea. The possibility that colchicine could block CFUs in the G₀ state or that it could interfere with the progress of CFUs through the G₁ and S phases of the cell cycle have been ruled out by experiments which demonstrated that colchicine (1 mg/kg b.w.), administered 10 min before hydroxyurea, does not reduce the number of CFUs triggered into the cell cycle as the consequence of hydroxyurea administration.     

Cultivation of fresh and frozen mouse bone marrow. - Application of methyl cellulose.

The culture of cells of both fresh and frozen mouse bone marrow on methyl cellulose (MC) was approached. We used 1.5%-concentration of MC and proved the stem cells of fresh and frozen mouse bone marrow to proliferate and form haemopoietic colonies on MC. We established that the freezing process did not significantly decrease the proliferative capacity of CFU-C stem cells.     

Homing and mobilization in the stem cell niche.

All mature blood cells are derived from the haemopoietic stem cell (HSC). In common with all other haemopoietic cells, stem cells are mobile, and it is this property of mobility that has allowed bone marrow transplantation to become a routine clinical option. Successful transplantation requires haemopoietic stem cells to home to the bone marrow, leave the peripheral circulation and become stabilized in regulatory niches in the extravascular space of the bone marrow cavity. This homing and tethering process is reversible - haemopoietic stem cells can be released from their bone marrow tethering through changes in molecular interactions, which are also important in homing following transplantation. The molecular mechanisms regulating this two-way flow of stem cells are beginning to be elucidated, and much recent data has emerged that sheds light on the processes and molecules involved in these complex physiological events. This article reviews current knowledge of the adhesive, homing and proliferative influences acting on HSCs and progenitor cells.     

Effect of bone morphogenetic protein-6 on haemopoietic stem cells and cytokine production in normal human bone marrow stroma

Normal human bone marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both bone morphogenetic protein (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term bone marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-10 per 10(5)cells in control cultures in a dose dependent manner to only 48+/-3 per 10(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on bone marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for bone repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term bone marrow culture.     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFU_c-GM compartment followed by an increase in the number of femoral CFU_s. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFU_e) compartment expanded to 168% of control, while the more differentiated (CFU_e) compartment was reduced (45% of control at day 28). Reductions in both ⁵⁹Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr × 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. the data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.     

Space flight effects on haemopoietic stem cells of the bone marrow of rats

Abstract. Changes in the number of haemopoietic stem cells (CFU-s) were studied in rats during the recovery day and selected post-recovery days after an 18–19-day flight on biosatellites Cosmos 936 and Cosmos 1129 . There was a decrease in the CFU-s number of the bone marrow of rats on the recovery day. On the 6th day post-recovery the CFU-s number was still depressed, while on the 25th day post-recovery it was elevated above control value. The differentiation ratio of transplanted bone marrow cells was not altered by space flight.     

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.     

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 cyclophosphmide 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 gamma-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.     

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.     

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.     

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.     

STEM CELL KINETICS IN THE L 5222 RAT LEUKAEMIA

The behaviour of normal haemopoietic stem cells of the bone marrow during the development of the acute transferable rat leukaemia, L 5222, has been investigated. The granulopoietic committed stem cells, measured by the in vitro colony technique, showed a marked decrease to less than half normal levels. Pluripotent stem cells included in the small lymphocytes of the bone marrow, and labelled with ³H-thymidine by the complete labelling method, showed only a modest decrease in number and an unchanged labelling intensity. The results suggest that in this leukaemia the pluripotent stem cells may be affected in such a way that they are unable to react by proliferation to the depletion of the succeeding cell compartments. This might be due to inhibition by leukaemic cells or to a disturbed feedback regulation between the committed and pluripotent stem cell compartments.     

Cryopreservation of human bone marrow cells by a modification of the two-step cooling technique

The first attempt to freeze human bone marrow cells with a two-step cooling method is reported. A simple and reliable way of obtaining stable first-step subzero freezing baths is described. One-milliliter samples each containing 20 × 10⁶ bone marrow cells and 10% Me₂SO were frozen in polypropylene cryotubes. Using these experimental conditions, the optimal freezing temperature was found to be in the range of −36 to 37.5 °C for BM progenitor cell (GM-CFC, CFU_E, and BFU_E) survival. Such temperatures were easily obtained in stable sludges of anisole or K₂CO₃ eutectic solution in water. The optimal holding time was 20 min before plunging tubes into liquid nitrogen. Similar or improved progenitor cell recoveries were observed compared with the conventional cooling technique. Adaptation of this two-step technique for the freezing of large volumes of BM cells for autografting is under investigation.