In vertebrate embryos, all hemopoietic tissues that are successively active during ontogeny (fetal liver, thymus, spleen and bone marrow) are colonized by extrinsic hemopoietic stem cells (HSC). The exception to this rule is the yolk sac (YS) whose progenitors arise in situ. Moreover, the YS is the first hemopoietic site to appear in the embryo. In consequence, it was considered as the primary source of the HSC that transfer from one site to the other until the demonstration that, in birds, the source of definitive HSC was located in the aortic region of the embryo. To assess a possible contribution of intraembryonic sites to mouse hemopoietic development, we developed an in vitro approach that permits the detection of multipotent hemopoietic progenitors in the region surrounding the dorsal aorta. This new site, the paraaortic splanchnopleura (P-Sp), is active from the 10 somite stage to the stage of fetal liver colonization by HSC. The in vitro analysis of the hemopoietic potential of the P-Sp revealed that: 1) progenitors present in this region are multipotent since a single micromanipulated P-Sp cell can give rise to mature B and T lymphocytes and various myeloid cells, when cultivated in appropriate conditions; 2) the P-Sp is the only intraembryonic site endowed with this potential; 3) numbering progenitors present in the YS and P-Sp indicated that precursors appear in both locations at the 10-12 somite stage reaching 15 in each site at 9.5 dpc (25 somites), shortly before the beginning of fetal liver colonisation. During a cytological study of the 9-11 dpc embryo aimed to disclose the cellular basis for our experimental results, two potential hemopoietic sites were uncovered. Cells clusters that resemble avian intraaortic hemopoietic clusters were found in the arteries located close to the coelomic cavity (dorsal aorta, umbilical and omphalomesenteric arteries). Moreover, groups of cells strikingly similar to YS blood islands were uncovered in the mesentery. All these sites are located in areas formed from the P-Sp and are fully developed at the onset of fetal liver colonization. These hemogenic sites most probably develop from the hemopoietic precursors present in the P-Sp.

In a search for assays that might facilitate identification of pluripotent stem cells with extended potentialities, we analysed the properties of hematopoietic progenitor cells detected in the extraembryonic yolk sac and in the intraembryonic part of human embryos between approximately 28 and 45 days of development. Cells from the yolk sac, the liver rudiment and the remainder of the embryo were plated in semi solid methylcellulose colony-assays supplemented with combinations of cytokines. Large BFU-E-derived colonies as well as granulocytic colonies were detected in every yolk sac sample. Interestingly, progenitor cells were also detected in the intraembryonic part, outside the liver and a subclass of these progenitors were detected that generated large granulomacrophagic colonies capable of generating secondary colonies when replated. These were preferentially located in the embryo. Colony-assays initiated with CD34+ cells sorted from the different tissues confirmed these data. These results first indicate that embryonic progenitors exhibit unique phenotypic features, and second, analysis of the distribution of progenitors between the different tissues may suggest the existence of other sites of hematopoietic production. More detailed analysis of the potentialities of these progenitors should now be assessed in vitro in cocultures assays and in vivo by reconstituting immunodeficient mice.

Immunohistochemistry was used to detect markers of the vascular, stromal and hematopoietic cell compartments in the human embryo and early fetus, from 3 to 15 weeks of gestation. CD34 expression was consistently observed at the surface of vascular endothelial cells from off earliest stages tested, at the single exception of embryonic liver blood vessels. Yolk sac hematopoiesis was very transient and limited to primitive erythropoiesis. Clusters of erythroblasts, monocytes and granulocytes appeared from 4 to 5 weeks of gestation in the liver rudiment. The early development of the bone marrow was marked by the rapid invasion, at 8 weeks, of long bone cartilaginous rudiments by CD68+ osteoclast precursors, CD34+ endothelial cells and by preosteoblasts, leading to the development of large vascular sinuses between ossifying trabeculae. Endogenous erythro- and granulopoiesis developed from week 11 in primary logettes always organized around an arteriole, in a loose stromal mesenchymal network established between the media of these arterioles and the sinusal endothelium. Round, hematopoietic CD34+ cells were seen occasionally in yolk sac blood vessels. In the liver they were rare and intermingled as single cells in the hepatocyte cords; strikingly, CD34+ hematopoietic cells could seldom be detected in the developing bone marrow. In contrast, compact clusters of non-endothelial, round CD34+ CD45+ hematopoietic cells were detected, during the 5th week of development, in close association with the ventral wall of the dorsal aorta. These cells exhibit phenotypic and functional characteristics of very primitive hematopoietic progenitors. This observation is in striking correlation with the evidence accumulated in animal models that stem cells for the late embryonic and adult hematopoietic systems develop inside the embryo per se, in the vicinity of the dorsal aorta. We thus suggest that these aorta-associated CD34+ cells, that exhibit an anatomic localization similar to that of the intraembryonic stem cells identified in the avian and murine embryo, are the real stem of human hematopoiesis.

Human cord and placental blood provides a rich source of hematopoietic stem cells. On the basis of this finding, umbilical cord blood stem cells have been used to reconstitute hematopoiesis in children with malignant and non malignant diseases after treatment with myeloablative doses of chemoradiotherapy. Early results show, that a single cord blood provides enough hematopoietic stem cells to provide short and long term engraftment, that the incidence and severity of graft versus host disease has been low even in HLA mismatched transplants. These results are encouraging enough, to embark on large scale banking of cord blood for purposes of future allogeneic and autologous stem cell transplantation, to promote studies on the unique properties of fetal and neonatal hematopoiesis, to study the immunological properties of cord blood cells, to initiate investigations on gene transfer into human cord blood cells for future gene therapy trials. This review will briefly summarize the current knowledge on cord blood transplantation as well as the future development of research on this unique source of hematopoietic stem cells.

Chimeras built between a quail embryo and a chick yolk sac have led to the demonstration that, in the avian model, intra-embryonic hemopoietic stem cells (HSC) relay HSC originating from the yolk sac. Converging experimental approaches have localized the emergence of intraembryonic HSC to the region of the aorta. Today's session will be dedicated to reviewing experimental data in mouse and man that clearly indicate that this model of hemopoietic ontogeny is also operating in mammals.

Differentiation of the hematopoietic tissue is controlled by growth factors which act precisely on stem cells arriving at a specific stage of differentiation. The recent identification of retinoic acid, a vitamin A metabolite, as an active differentiating agent of acute promyelocytic leukemia, has allowed to define a normal group of growth and differentiation factors of the myeloid tissue: vitamins A and B and thyroid hormones.

Autologous bone marrow transplantation for the treatment of solid tumors in adults remains an uncommon therapeutic approach. The feasibility of such high-dose therapies is clearly proved, especially with the advent of hematopoietic growth factors and the rescue by the peripheral stem cells to reduce the duration of the chemotherapy-induced myeloid aplasia. The question is to exactly define the place of high-dose therapy in the land of solid tumors. For the treatment of primary chemoresistant gonadal germ-cell tumors, the possibility to cure the patients and the interest of high-dose therapy with autologous bone marrow transplantation are clearly demonstrated. As consolidation for the treatment of poor prognosis tumors, the place of high-dose therapies remains moot. For the treatment of chemoresistant extragonadal germ-cell tumors, especially for primary mediastinal tumors, the level of resistance to cisplatin-based chemotherapy regimens is generally too high to be overcome by intensive therapies given as single course or as tandem courses. However in association with debulking surgery, this therapeutic approach has to be considered for some patients. In the treatment of poor prognosis breast cancer, high-dose therapy with autologous bone marrow transplantation or with peripheral stem cells support is able to convert some patients with partial response into complete responders. However, the consequences on overall survival and on disease-free survival are not evident. For metastatic breast cancer and for poor-prognosis tumors (inflammatory breast cancer, axillary metastatic nodes > or = 8), the interest of high-dose therapy has to be determined by randomized studies. These studies are ongoing in USA and in Europe. For the treatment of poor-prognosis ovarian cancer, the situation is more difficult to appraise. Once again, randomized studies have to be done to precisely define the place of high-dose therapy. In the land of small-cell lung carcinomas, high-dose therapy is actually forsaken by most of authors, even for limited diseases. The results of previous studies are disappointing. Moreover, occult medullary micrometastases involvement is frequent, once again even in limited diseases. However new therapeutic associations, as the ICE regimen (IFM, Carboplatin, VP-16) delivered as single or tandem therapy, have to be studied, especially as early consolidation therapy for the treatment of limited small-cell lung carcinomas.

The CFU-GM (granulo-macrophagic colony forming unit) content of 130 bone marrow samples and 105 cytapheresis blood samples was evaluated by two culture techniques, one in agar with human placenta conditioned medium as stimulating factor, the second in methyl cellulose with a cocktail of recombinant G-CSF, GM-CSF, IL-3 and erythropoietin as stimulating factors. The aim of the study was to evaluate in methyl cellulose the CFU-GM threshold doses necessary to ensure hemopoietic reconstitution in autologous transplantation that we previously determined in the agar technique. Thirty-one out of the 130 BM samples were also tested after incubation with mafosfamide, and 33 after freezing and thawing. Results showed that the numbers of CFU-GM in the 2 techniques were significantly correlated (p < 0.0001). The strongest correlation was found with the formula "log CFU-GM methyl = a log CFU-GM agar + b" with a slight variation in the values of a and b among the 4 settings, i.e. unmanipulated BM, mafosfamide-treated BM, frozen-thawed BM and unmanipulated blood samples. According to the formulas, the threshold doses of CFU-GM for BM and blood, previously determined in the agar technique (respectively 10(4)/kg and 5 x 10(4)/kg) were calculated for CFU-GM grown in methyl and corresponded respectively to 2.3 x 10(4)/kg and 2.1 x 10(5)/kg.

Peripheral blood stem cell (PBSC) transplantation is now widely used as technics of stem cell transplantation. If it is efficacious in terms of short-term engraftment, many questions remain unanswered: which is the best technic of mobilization, what role for growth factors after graft, which benefit to add bone marrow stem cells? So, comparative trials are necessary to compare bone marrow transplantation and peripheral stem cell transplantation in terms of long-term engraftment and anti-tumoral effect. However, the present main point is to better define the adequate estimation of circulating hematopoietic progenitors according to the technics of collection: evaluation of CFU-GM and CD34+ levels are inadequate for estimation of immature progenitor cells which vary a lot according to the types of chemotherapy or of growth factors used for mobilization.

It is now possible to achieve prolonged remission of malignant lymphoma and certain cancers with high-dose chemotherapy followed by autograft with haematopoietic stem cells. We tested such a protocol, evaluating haematologic recovery, in order to determine the total cost of hospitalization.

A balance of both stimulatory and inhibitory factors interfere with gross and precise hematopoiesis regulation. A number of inhibitory molecules may endeavour a physiological role (cytokines like TGF beta, TNF alpha, platelet factor 4 PF4, Interferons, MIP1 alpha or peptides like p-EEDCK, Ac-SDKP. Hemopoietic primitive stem cells are relatively spared from chemo- or radiotherapy toxicity when they stay in a quiescent G0 state. They suffer from repeated toxic treatments when they undergo cycling. Molecules like the AcSDKP (seraspenide) inhibit in a reversible way stem cell and progenitors cycling, affording them a significant degree of protection from chemotherapy (cytarabine, cyclophosphamide, 5FU, AZT) and ionizing radiation toxicity. A clinical phase Ib study suggests protective effects from cytarabine toxicity.

Novel therapeutic agents in the form of recombinant human hematopoïetic cytokines have been developed. Two of them granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage factor (Gm-CSF) have become widely available commercially. A wholly new technique of supportive care became available to hematologists for patients from whom myelosuppressive chemotherapy represents the optimal treatment. These agents moved directly into large-scale prospective, randomized placebo-controlled studies. The data in support of glycosylated G-CSF as an adjunct to aggressive chemotherapy (ACVB) in lymphoma were provided by a randomized study on 162 patients. Reduction of duration of neutropenia < 500/microliter from 4 day to 1 day was observed during four cycles, as well as the reduction of the duration of infection and time in hospital. The same effects have been described with Gm-CSF and less intensive regimens. Hematopoietic growth factors are accepted as accelerating hematologic recovery after bone marrow grafting. In our randomized study with G-CSF, 315 patients were included. Neutrophil recovery > 1,000/microliter for 3 days was seen earlier in G-CSF treated patients (16 vs 27 d). Time to neutrophil > 500/microliter was reduced (14 vs 20 d). The difference was significant both in autograft and allograft patients. Patients had fewer days of infection and spent less time in hospital. In all randomized studies with G or Gm-CSF no difference in survival was observed at one year. If the only benefit of cytokines is to diminish toxicity, the next goals will be to determine the most appropriate situation in which these agents are most justified and to use these agents in novel approaches. Mobilization of peripheral blood stem cell with hematopoietic factors alone or in association with chemotherapy allows a further improvement in controlling toxicity after transplantation with a median time to neutrophil and platelets recovery of 12 days. Stem cells transplantation will become more easily integrated in dose-intensive treatment.

Hematopoiesis is the cellular system which leads to the continuous production of blood cells. This highly complex cellular system is organized into three main compartments: (i) stem cells which are both pluripotent and theoretically capable of self renewal; (ii) hematopoietic progenitors which are committed to (only) one cell lineage and are able to proliferate along each particular differentiation pathway; (iii) a maturation compartment in which cells become morphologically identifiable since they synthesize lineage specific proteins. The maturation cell compartment represents the majority of marrow cells. At the present time, the regulation of true stem cells remains poorly understood since these cells are difficult to assay in vitro. In contrast, the regulation of each hematopoietic lineages becomes to be well known. These knowledges are mainly due to two reasons: (i) hematopoietic progenitors can be purified and assayed in culture. Their proliferation and differentiation are strictly dependent upon the presence of hematopoietic growth factors; (ii) these different hematopoietic growth factors have been isolated and their cDNA cloned. Erythropoiesis and megakaryocytopoiesis are two branches of hematopoiesis which lead to the production of RBC and platelets, respectively. These two cell lineages have several common features. However, they markedly differ by their regulation since RBC production depends upon one main stimulus (hypoxia) and, therefore, the terminal erythroid differentiation is regulated by a single growth factor. In contrast, regulation of platelet production may depend on several stimuli such as the platelet mass (homeostasis), inflammation, infection and hypoxia. Therefore, several cytokines are involved in the regulation of megakaryocytopoiesis. In addition, the mechanisms of platelet production are highly complex and, in contrast to all the other hematopoietic lineages where the production of mature cells depends on a single parameter (the proliferation during differentiation), three independent parameters modify thrombopoiesis: a) the number of marrow megakaryocytes (MK) (proliferation of the precursor cells). b) the megakaryocyte volume which directly depends on the MK ploidy. During MK differentiation, MK precursors switch from a mitotic process (DNA duplication followed by cytokinesis) to an endomitotic process (DNA duplication without cytokinesis). Endomitosis is a specific process of the megakaryocytic differentiation and differs from all the other cellular models of polyploidization by the existence of a single polyploid and polylobulated nucleus in each cell. This polyploidization induces a major amplification of the platelet production since it is associated with a parallel increase in the cytoplasmic mass.(ABSTRACT TRUNCATED AT 400 WORDS)

The healing of a wound or blood cell regeneration following a hemorrhage illustrates the existence of strong homeostatic mechanisms. The numbers of cells are maintained constant through a powerful control system of cell proliferation in the skin and bone marrow. For example, the control mechanisms were not understood at the beginning of the century and the first evidence of a humoral factor was reported in 1905 by P. Carnot and Deflandre. They found that after bleeding a rabbit, there was in its serum 24 hours later some factor which stimulated red cell production in normal rabbits. They called it hemopoietin, but it was later called erythropoietin. During the past decades, abscopal effects strongly supported the role of humoral factors. Further progress has been made. The first was the description of the growth factor cell receptor system by S. Cohen and R. Levy-Montalcini. This system which was initially described for skin is also valid for hemopoiesis and lymphopoiesis. Another major progress was the concept of hemopoietic stem cells. The first control mechanisms which regulate proliferation and differentiation in hematopoietic tissue were elucidated less than 25 years ago. Enormous progress has since been made to enable the purification and identification of several growth and inhibitory factors which already have their place in daily clinical practice. Their clinical role will continue to grow and a better understanding of these mechanisms will make a fundamental contribution to cellular biology.

Legitimate efforts to reduce health care costs, especially of intensive protocols including transplantation for haematological diseases, require realistic economic evaluations. We determined the direct cost of intensive chemotherapy associated with total body irradiation and autologous blood stem cell transplantation in patients with multiple myeloma.

Spontaneous recovery from severe traumatic lesions of the central nervous system (CNS) does not occur in adult mammals for two main reasons: the lost neurons are not replaced and the surviving axotomized nerve cells do not regenerate the missing part of their cut axon, thus failing to reestablish the original anatomical and functional connections. The likeliest explanation for this lack of axonal regrowth is powerful inhibitory effects from mature reactive astrocytes and oligodendrocytes. However, experimental strategies making use of transplantation techniques have proved to be rather efficient. Thus, with regard to spinal cord injury: 1) lost spinal neurons (especially motoneurons) can, to some extent, be replaced by foetal spinal neurons transplanted to the lesion site; 2) irreversible damage to spinal projections from supraspinal neurons (mainly localized in the brain stem) can partially be compensated by grafting homologous foetal nerve cells caudally with regard to the spinal lesion site; 3) axogenesis from transplanted nerve cells or axonal regrowth from injured host neurons can be triggered and guided towards central or peripheral targets by autologous peripheral nerve segments which bypass the lesion site. This efficient cellular therapy is on the way to be complemented by a gene therapy that will allow the introduction, into the brain and the spinal cord, either of genetically modified cells from various origins, or of selected beneficial genes previously integrated into viral vectors.

Cytogenic analysis of leukemic cells has proven to be a mandatory part of the diagnosis of malignant hemopathies. Recurring clonal cytogenetic abnormalities may be divided into those exclusively associated with myeloid disorders, those uniquely observed in lymphoid diseases, and those detected in both myeloid and lymphoid hemopathies. Several of the common defects are characteristic of specific FAB types or subtypes and are associated with specific clinico pathologic syndromes and clinical complications. Cytogenetic abnormalities have served to define relatively homogeneous subsets of malignant hemopathies which are not evident from morphological and other available markers. Cytogenetic findings have been demonstrated to be powerful indicators in predicting clinical course and outcome in patients and in guiding their management. Given the significant progress made in the treatment of malignant hemopathies, it is very important to identify parameters which may be used to predict whether patients will respond favorably to standard therapies or if they are unlikely to do so and require alternative strategies, such as bone marrow transplantation. Cytogenetic studies have also provided important insights into the understanding of malignant transformation processes. In a number of recurring chromosome translocations characteristic of leukemias and lymphomas the genes that are located at the breakpoints have been identified. Molecular analysis has revealed that alteration in expression of these genes or in the properties of the encoded proteins resulting from the rearrangements plays an integral part in malignant transformation. Studies of clonality have suggested that several chromosome abnormalities may arise in pluripotent hemopoietic stem cells, whereas others may originate in cells of more restricted lineage. The author focuses first on the implications of the karyotype in the diagnosis and the prognosis of myeloproliferative syndromes, acute leukemias and myelodysplastic syndromes, then on the interest of describing new clinical-cytogenetic associations. Finally, some of the recent results obtained in a cytogenetic study of myelodysplastic syndromes are discussed.

In the late seventies, iodinated contrast agents (ICA) were considered to be a major cause of acute iatrogenic renal failure. Over the last decade new contrast agents have been synthesized, nonionic and less hyperosmolar. Analysis of 25 series published between 1975 and 1989 does not allow precise evaluation of the incidence of acute renal failure due to ICAs. This incidence varies from 3.7 to 70% of cases according to the series, with an average figure of 10.2%. Such discrepancies stem from different definitions of renal failure, as well as varying criteria adopted for defining "nephrotoxicity", high-risk patients and the better safety of new, nonionic and less hyperosmolar agents. The pathophysiology of ICA nephrotoxicity was mainly studied in laboratory animal models. Three main factors are involved in an inducing ICA-mediated decrease in glomerular filtration rate: reduction of the renal plasma flow, a direct cytotoxic effect on renal tubular cells and erythrocyte alteration leading to intrarenal sludge. Excluding dysglobulinemias with urinary excretion of immunoglobulin light chains, which represent a special case of maximum nephrotoxicity, 4 main risk factors of renal toxicity have been identified in nondiabetic subjects: previous renal failure with serum creatinine levels greater than 140 mumol per liter, extracellular dehydration, age over 60 and use of high doses of ICA and/or repeated ICA injections before serum creatinine levels return to baseline. Diabetes does not seem to represent a serious risk factor, except in patients with diabetic glomerulosclerosis. Ongoing treatment with nonsteroidal antiinflammatory drugs (NSAID) potentiates the hazards of renal failure. Preventive measures for avoiding ICA nephrotoxicity are threefold: maintain or restore adequate hydration with saline infusion, stop NSAID treatment several days before ICA administration, and allow a 5 day interval before repeating contrast media injections. New, nonionic and moderately hyperosmolar contrast agents appear to be much less nephrotoxic than conventional ICAs in laboratory animals and in high-risk patients. It is advisable to select such contrast media for investigating high-risk patients. This approach was recently substantiated in well designed, randomized clinical studies which included more than 2,000 patients.