Functional, molecular and proteomic characterisation of bone marrow mesenchymal stem cells in rheumatoid arthritis.

OBJECTIVE: Bone marrow (BM) mesenchymal stem cells (MSCs) are being considered as potential therapeutic agents in various inflammatory autoimmune diseases for their tissue-repair and anti-inflammatory tissue-protective properties. This study investigates the reserves and function, the molecular and proteomic profile and the differentiation potential of BM MSCs in patients with active rheumatoid arthritis (RA). METHODS: We evaluated the frequency of MSCs in the BM mononuclear cell fraction using a limiting dilution assay, the proliferative/clonogenic potential and the capacity of cells to differentiate towards the osteogenic/chondrogenic/adipogenic lineages using appropriate culture conditions. We also assessed the molecular and proteomic characteristics in terms of inflammatory cytokine gene and protein expression, the relative telomere length and the survival characteristics of BM MSCs. RESULTS: MSCs from patients with RA (n = 26) and age- and sex-matched healthy individuals (n = 21) were similar in frequency, differentiation potential, survival, immunophenotypic characteristics, and protein profile. Patient MSCs, however, had impaired clonogenic and proliferative potential in association with premature telomere length loss. Transcriptome analysis revealed differential expression of genes related to cell adhesion processes and cell cycle progression beyond the G1 phase. Previous treatment with methotrexate, corticosteroids, anti-cytokine and biological agents or other disease-modifying anti-inflammatory drugs did not correlate with the clonogenic and proliferative impairment of BM MSCs. CONCLUSION: In spite of some restrictions related to the impaired clonogenic and proliferative potential, our findings support the use of autologous BM MSCs in RA and may have important implications for the ongoing efforts to repair tissue injury commonly seen in the course of the disease.

Properties and potential of bone marrow mesenchymal stromal cells from children with hematologic diseases.

BACKGROUND: Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases. METHODS: BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed. RESULTS: MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone. DISCUSSION: MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

A sub-population of high proliferative potential-quiescent human mesenchymal stem cells is under the reversible control of interferon alpha/beta.

Type I interferon (IFN) is shown to control the reversible quiescence of a primitive human bone marrow mesenchymal stem cell (MSC) subpopulation. A 24 h pre-treatment of Stro1+/GlycoA- or CD45-/GlycoA- subpopulations with a monoclonal antibody (mAb) against the IFNAR1 chain of the human type I IFN receptor (64G12), or with a polyclonal anti-IFNalpha antibody, resulted in a marked increase in the number of very large colonies (CFU-F >3000 cells) obtained in the presence of low, but necessary, concentrations of bFGF. Over a 2-month culture period, this short activation promoted a faster and greater amplification of mesenchymal progenitors for adipocytes and osteoblasts. Activation correlated with inhibition of STAT1 and STAT2 phosphorylation and of STAT1 nuclear translocation. A non-neutralizing anti-IFNAR1 mAb was ineffective. We demonstrate that control and activated MSCs express ST3GAL3, a sialyltransferase necessary to produce the embryonic antigens SSEA-3 and -4. Interestingly, activated MSC progeny expressed SSEA-3 and -4 at a higher level than control cultures, but this was not correlated with a significant expression of other embryonic markers. As MSCs represent an essential tool in tissue regeneration, the use of 64G12, which rapidly recruits a higher number of primitive cells, might increase amplification safety for cell therapy.

The detection of colony-stimulating factors and steel factor in adherent layers of human long-term marrow cultures using reverse-transcriptase polymerase chain reaction.

In human long-term marrow cultures granulomonopoiesis is maintained for several weeks. Studies on granulomonocytic progenitors (CFU-GM) and their progeny have shown that survival, proliferation, differentiation and maturation of these cells are controlled by a set of glycoproteins, the colony-stimulating factors (CSFs) and the Steel factor. We have studied the expression of these factors using reverse transcriptase polymerase chain reaction (RT-PCR) in 17 adherent layers of normal bone marrow at 3, 5 or 7 weeks of culture. We have taken the 5637 bladder carcinoma cell line as a control for expression of GM-CSF, M-CSF, G-CSF and Steel factor, and PHA-activated T lymphocytes as a control for expression of multi-CSF (interleukin 3, IL-3). We have found that GM-CSF was expressed in the 17 adherent layers without induction by interleukin 1 beta (IL-1 beta). M-CSF was also detected in all cases, but in two early-stage (week 3 and week 5) cultures only after stimulation by IL-1 beta. G-CSF was detected in only 11 cases (three without IL-1 beta, and eight after addition of IL-1 beta). Steel factor was detected in 14 cases (ten without IL-1 beta, and four after addition of IL-1 beta). IL-3 was not detected even by means of nested RT-PCR. These data indicate in six late-stage (week 5 or week 7) cultures G-CSF messenger concentrations 10(3)-fold less than in 5637 control cells (for an identical amount of total cellular RNA). A similar conclusion may be drawn for Steel factor in three late-stage cultures. For IL-3 our negative results indicate a messenger concentration 10(5)-fold less than in activated T lymphocytes. These results suggest a crucial role for GM-CSF and M-CSF in the maintenance of granulomonopoiesis in human long-term cultures. The role of G-CSF and Steel factor may be more marginal. Eventually IL-2 may not be involved in the regulatory process.

Effects of cryopreservation on the adherent layer of human long-term bone marrow cultures: study of cell phenotypes.

This report describes the effects of cryopreservation on the adherent layer of human long-term bone marrow cultures (HLTBMC). Stromal cells are believed to be the most important cells of medullar microenvironment to regulate hematopoiesis. To study effects of cryopreservation, we compared the cell phenotypes of adherent layers of fresh and frozen-thawed bone marrows. To characterize stromal cells we used monoclonal antibodies reacting with components of these cells (CGA-7 alpha SM and gamma SM actin isoforms; HHF-35, all muscle actin isoforms; BMS-1, stromal cell lysosomes). The other components studied were: fibronectin (BMS-2 monoclonal antibody) and hematopoietic cells (monoclonal antibodies against CD45, CD33, and CD14). Results show a decrease of cells positive for CGA-7, HHF-35, and BMS-1, in adherent layer of HLTBMC of frozen-thawed bone marrows. Expression of BMS-2 is unchanged, and CD45 and CD14-positive cells proportionately increased. These results are consistent with an impairment of stromal cell proliferation in frozen-thawed marrows, without impairment of most stromal cell functions. The difference between stromal cell and hematopoietic cell kinetics seems to be an additional fact suggesting a different origin for both cell populations.

Nontransformed colony-derived stromal cell lines from normal human marrows. II. Phenotypic characterization and differentiation pathway.

In a previous report, we described a method to generate cell lines derived from stromal colonies (colony-derived cell lines [CDCL]). In a first step, colonies were obtained from plating cells from adherent layers of human long-term bone marrow cultures (LTBMC) in methyl-cellulose in the presence of interleukin-1 beta (IL-1 beta) (20 U/mL) and tumor necrosis factor-alpha (TNF-alpha) (200 U/mL). In a second step, cell lines were derived from individual colonies cultured in liquid medium with 20 ng/mL basic fibroblast growth factor (bFGF). In this report, we describe the phenotype of cells from more than 100 cell lines. CDCL did not contain cells of hematopoietic origin, which indicates that the culture system did not allow the growth of hematopoietic precursors. CDCL did not contain endothelial-like cells, similarly to primary adherent layers. CDCL comprised stromal cells, as defined by membrane antigens recognized by monoclonal antibodies 6-19, Stro-1 and 1B10. These cells belonged to the family of connective tissue-forming cells since they synthesized interstitial collagens I, III, and V; nonplasmatic EDa+ and EDb- fibronectin; tenascin; and chondroitin-sulfate. Study of the time course of CDCL showed that the lines differed from one another according to their proliferative capacity: 32% of CDCL grew quickly, yielding about 10,000 cells in 10 days; 36% of CDCL grew slowly, yielding 1000 cells in 10 days; and the remaining 32% had intermediate proliferative capacity. For CDCL with a high proliferative capacity, a distinctive differentiation pattern could be described. At culture, inception cells from the lines were vimentin and laminin. Over time, several cytoskeletal and extracellular matrix (ECM) proteins indicative of vascular smooth muscle differentiation were expressed, including: the alpha-SM-actin isoform; the actin-binding proteins, smooth muscle myosin-heavy chain (SMMHC), SM1; h-caldesmon; calponin; gelsolin; and the ECM proteins, collagen IV and elastin. In full-grown lines, cells were similar to immature, intimal, vascular smooth muscle cells as found beneath the endothelium in adult aortas. Because of the coupling between proliferation and differentiation, the differentiation pattern seems to be under genetic control. However, since the coupling was not stringent during the whole lifespan of the lines, it is possible that cytokines are also involved, ensuring autocrine regulation of CDCL development.

Nontransformed colony-derived stromal cell lines from normal human marrows. III. The maintenance of hematopoiesis from CD34+ cell populations.

Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth-muscle cell repertoire. Here we study the maintenance of hematopoiesis by this cell population. We show that CDCLs allow the generation for several weeks of stroma-adherent colonies (comprising a cobblestone area) from CD34+, CD34+/CD38+, and CD34+/CD38- cells. Stroma-adherent colony-forming cells (CFCs) from CD34+/CD38- cells reach a maximum at week 4 and limiting dilution analysis gives a frequency of 1 per 10 cells seeded; in contrast to this, CFCs from CD34+/CD38+ cells are optimal by week 2 and the frequency is then only 1 per 120 cells seeded. Stroma-adherent colonies comprise hematopoietic cells from all lineages except the T lymphocytic, with a majority of granulomonocytes. CDCLs also allow the amplification of granulomonocytic colony-forming units (CFU-GMs), since cumulative outputs of CFU-GMs by week 6 are 190 and 8 times that observed at culture inception for the CD34+/CD38- and CD34+/CD38+ cell populations, respectively. Our results suggest that stromal cells from CDCLs allow the maintenance of primitive hematopoietic precursors and induce their proliferation and differentiation. This study underscores the potential role of one of the microenvironmental cell populations, that of myoid cells, in the regulation of hematopoietic precursor behavior.

Toward cord blood banking: density-separation and cryopreservation of cord blood progenitors.

It has been shown that cord blood collected at birth can be used to successfully engraft a human lymphocyte antigen (HLA)-matched sibling suffering from a malignant disease. It has been further suggested that this source of cells may be used in unrelated but HLA-compatible patients. These wide indications would imply the establishment of cord blood banks comprising 10(5) or more samples. In this report we show that it is possible to fractionate and freeze cord blood samples without major loss in granulomonocytic or erythroblastic progenitors (CFU-GM and BFU-E). Density separation should be carried out using Percoll of density 1.080. Separated samples should be frozen and thawed in the presence of DNase I. This procedure should allow the storage of approximately 10 mL samples in cryotubes containing a number of CFU-GM and BFU-E sufficient to engraft a patient weighing less than 30 kg. These data provide a rationale for establishing cord blood banks.

Human granulocyte colony growth: differences between serum-free and serum-dependent cultures.

Human granulocyte colony formation has been observed in serum-free methylcellulose cultures with Iscove medium, delipidated bovine serum albumin, iron-saturated transferrin, alpha-thioglycerol, oleylpalmitoyl lecithin, cholesterol, and linoleic acid using serum-free human placental-conditioned medium (SF-HPCM) as the source of colony stimulating factor (CSF). Dose-response curves for SF-HPCM indicated a lower sensitivity to colony-stimulating activity in serum-free cultures than in serum-dependent cultures. Gel filtration of SF-HPCM revealed that CSF fractions with molecular weights in the range of 30 kD are inefficient in serum-free cultures, while fractions with molecular weights in the range of 40 kD stimulate granulocyte colony formation in both types of cultures. These results demonstrate that serum constituents modulate the effects of one of the stimulating factors for granulocyte colony formation, and that serum-free culture conditions are essential for establishing the growth factor requirements of the granulocyte lineage.

Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

Production and consumption of the tetrapeptide AcSDKP, a negative regulator of hematopoietic stem cells, by hematopoietic microenvironmental cells.

This study was performed to evaluate the role of human microenvironmental cells in the metabolism of AcSDKP, a physiological inhibitor of hematopoietic stem cells. Using long-term marrow cultures (LTMCs), whose medium already contained a baseline value of AcSDKP, we found after 2 weeks a net output in the culture supernatant indicating that release by cells from the adherent layer was superior to consumption of the peptide. Since human microenvironmental cells consist of macrophages and vascular smooth-muscle-like stromal cells we generated pure populations of macrophages (by culturing cord blood cells in the presence of granulomonocytic colony-stimulating factor) and of stromal cells (generated by stromal colonies). We found in supernatants of macrophage cultures a significantly (p < 0.01) increased level of AcSDKP (compared with value in medium) while in supernatants of stromal cell cultures the level was decreased. Cell content of angiotensin-converting enzyme (ACE) in stromal cells was higher than in macrophages, which suggests a degradation of AcSDKP by stromal cells because of their higher amount of ACE. Finally, we analyzed the content of AcSDKP in adherent layers of LTMCs (with or without extracellular matrix [ECM] components), macrophages, and stromal cells. We found levels of AcSDKP of 1.5 pMol per 106 cells in extracts from macrophages or from stromal cells. On the contrary, extracts from primary layers of LTMCs contained 3 times more AcSDKP; however, after treatment of primary layers by collagenase, AcSDKP level fell to 1 pMol per 10(6) cells. Immunofluorescence using an anti-AcSDKP monoclonal antibody showed an extracellular network in certain areas of LTMCs. This study shows that 1) macrophages synthesize and release in the supernatant AcSDKP, 2) stromal cells probably degrade the peptide via ACE, and 3) components of the ECM from LTMCs serve as a reservoir for the peptide. These results are reminiscent of what has been described for growth factors, produced by microenvironmental cells, and stored in the ECM in close vicinity to hematopoietic precursors.

The cytoskeleton of stromal cells from human bone marrow cultures resembles that of cultured smooth muscle cells.

The cytoskeleton of stromal cells from the adherent layer of human Dexter-type cultures has been studied. It was found that the stress fibers contained actin specific for smooth muscle, mainly the alpha SM actin isoform. The intermediate filaments consisted of vimentin, and there were no desmin filaments. This pattern was similar to that of cultured vascular smooth muscle cells. The detectability of the alpha SM actin isoform is coincident with the appearance of stromal cells in long-term marrow cultures and may provide a useful marker for stromal cells. The potential in vivo cellular counterpart for stromal cells generated in the Dexter-type culture system is discussed.

Nontransformed colony-derived stromal cell lines from normal human marrows. I. Growth requirement and myelopoiesis supportive ability.

We report a new method for generating nontransformed human stromal cell lines with a replicative potential of 20 to 25 doublings yielding 10(6) to 3 x 10(7) cells after 4 to 6 weeks. Cells from week-3 to -6 adherent layers of human long-term bone marrow cultures (LTBMC) were plated in methylcellulose in the presence of 20 U/mL interleukin-1 beta (IL-1 beta) and 200 U/mL tumor necrosis factor-alpha (TNF-alpha). After 2 to 3 weeks, we obtained 180 +/- 14 colonies per 10(5) cells seeded. These well-delineated colonies with a dense central core consisted of up to several hundred tightly packed, identical, large refractile cells. Colonies were determined to be clones by sequential examination of the cultures and the linear relationship between the number of colonies counted and cells seeded. Colony-derived cell lines (CDCL) were developed by seeding individual colonies in long-term culture medium (LTCM) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF). The selection of colonies yielding lines with high proliferative capacity was due to the presence of IL-1 beta and TNF-alpha in the semisolid medium. The most effective concentrations for clonal selection were 200 U/mL TNF-alpha and 20 U/mL IL-1 beta. The growth of CDCL in liquid culture depended on the presence of bFGF, with the most effective concentration at 20 ng/mL. CDCL were able to maintain the output of colony-forming units granulocyte/macrophage and burst-forming unit-erythrocyte (CFU-GM and BFU-E) for several weeks from cocultured CD34+ marrow cells. The weekly CFU-GM and BFU-E output from weeks 2-5 was at least the same as observed when using passaged adherent layers. CDCL represent a progenitor cell population for stromal cells that may prove a suitable model for the study of the relationship between marrow stromal cells and hematopoiesis.

Distribution of surface-membrane molecules on bone marrow and cord blood CD34+ hematopoietic cells.

We have investigated the distribution of membrane molecules on CD34+ hematopoietic cells isolated from human bone marrow (BM) and cord blood (CB). A distinct CD10+ population was present in BM, but it was not detected in CB. Most CD34+ CD10+ cells in BM were B-cell precursors (BCP), because they expressed CD19. However, CD40 and CD37 were found on the majority of CD34+ cells from either BM or CB, demonstrating that these antigens are not restricted to B-lineage CD34+ cells. CD40 and CD37 were lost during culture of CD34+ cells in the presence of interleukin 3 (IL-3), indicating transient expression early in myeloid development. CD13 antigen was detected on virtually all CD34+ cells from BM and CB. Accordingly, CD13 was present on CD34+ CD10+ cells, demonstrating that this structure is not restricted to myeloid CD34+ cells. In contrast, myeloid CD33 antigen was not detected on CD34+ CD10+ cells. Expression levels of CD13 and of CD33 were heterogeneous in BM, reflecting diversity within the resident CD34+ population. CD25 and CD71 were found on a proportion of CD34+ cells from either BM or CB and maintained during culture in IL-3, consistent with a distribution on activated cells. Finally, a variety of adhesion receptors were present on CD34+ cells. These included the alpha 4 beta 1 (VLA-4), alpha 5 beta 1 (VLA-5), and alpha L beta 2 (LFA-1) integrins, as well as ICAM-1, LFA-3, H-CAM, and LAM-1. Expression of adhesion receptors was remarkably similar in BM and CB, and it followed an all-or-nothing pattern that failed to delineate CD34+ subsets. Taken together, our data show that although CD34+ cells from BM constitute a more heterogeneous population, resident and circulating CD34+ cells largely display the same cell-surface molecules.

Vascular smooth muscle differentiation of murine stroma: a sequential model.

Previous studies by our group showed that stromal cells from human long-term marrow cultures were mesenchymal cells following a vascular smooth muscle pathway. The present study using 58 immortalized stromal lines from different hematopoietic sites was conducted to verify whether this hypothesis also held true for murine stroma. Principal components analysis performed using cytoskeletal and extracellular matrix proteins allowed the segregation of five factors explaining more than 70% of the variance. Factor I, including osteopontin and vimentin, and factor II, laminins and fibronectins, were representative of the mesenchyme. The remaining three factors were representative of vascular smooth muscle: factor III, including alphaSM actin, SM alpha actinin, SM22alpha, EDa+ fibronectin, and thrombospondin-1; factor IV, metavinculin and h-caldesmon; and factor V, smooth muscle myosin SM1 and desmin. All lines expressed factors I and II; 53 lines expressed factor III, 35 lines expressed factor IV; and 11 lines expressed factor V. A second principal components analysis including membrane antigens indicated the cosegregration of vascular cell adhesion molecule-1 with osteopontin and that of Ly6A/E with vimentin, whereas CD34 and Thy-1 appeared to be independent factors. The heterogeneity of vascular smooth muscle markers expression suggests that harmonious maintenance of hematopoiesis depends on the cooperation between different stromal cell clones.

Relationship between thyrotropin stimulation and radioiodine uptake in lung metastases of differentiated thyroid carcinoma.

To examine whether the injection of bovine TSH (bTSH) produces maximal radioactive iodine uptake (RAIU) in lung metastases in patients with differentiated thyroid cancer, 10 patients were studied 12 times. In 10 of these studies, an initial RAIU measurement was performed immediately after 3 injections of 10 IU bTSH given immediately after T3 withdrawal. Another RAIU measurement was performed 7-19 days after T3 withdrawal. Uptake increased in all patients even when it was clearly detectable immediately after bTSH stimulation. Thus, 3 days of bTSH stimulation in these patients did not lead to maximal 131I uptake, and it could only be reached after prolonged endogenous TSH stimulation. bTSH was not injected in the 2 other patients, in whom 6 RAIU measurements were carried out. Radioiodine uptake increased with time in both patients. It appears that both the level of endogenous TSH and the length of stimulation play a determining role in RAIU. This might explain why 3 days of bTSH stimulation are insufficient to elicit maximal 131I uptake.

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission.

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.

Granulomonocytic colony forming cells in myelofibrosis: concentrations within hepatic blood and peripheral blood.

Hepatic and peripheral blood GM-CFC concentrations were compared in 8 cases of myelofibrosis. Hepatic blood concentrations were significantly higher (p less than 0.05). The density-distribution profile for hepatic blood GM-CFCs shifted to the left as compared to peripheral blood in 4 cases out of 5. The maximal blood transit time for circulating GM-CFCs was about 1 min. These results suggest that spleen and liver are a production site of blood GM-CFCs in myelofibrosis. In particular, they would be responsible for the presence in blood of the lightest GM-CFCs (less than 1.060 g cm-3). Increased GM-CFC inflow from bone marrow and/or liver and spleen to blood is the single explanation for the increased value of the circulating GM-CFCs.

Effects of treatment of malignant lymphomas on the granulocytic progenitor cells (CFUc).

The goal of this work was to appraise the after-effects of radiotherapy and associated radio- and chemotherapy on hematosarcomas by studying the granulocytic progenitor cells (CFUc). The bone marrow of 14 subjects was cultured in methylcellulose 1--6.5 years after completion of radiotherapy. The results were recorded as the number of CFUc per 10(5) nucleated medullary cells plated and per 10(5) metamyelocytes plated, and the total number of medullary CFUc was calculated. These different modes of expression are discussed. The post-radiotherapeutic effects appeared to be closely correlated with the volum irradiated. Chemotherapy seemed in several cases to accentuate the effects. Nevertheless, the organism can operate a powerful compensatory mechanism by means of increased mitosis, and in addition, certain subjects are able to maintain a normal number of CFUc after an aggressive treatment. It was possible in certain cases to study the medullary regeneration of the irradiated zone by culture of the CFUc and by the 59Fe external counting. Its extent after radiotherapy seemed for both the granulocytic and the red cell line to be correlated with the volume irradiated. Subject to the study of new cases, regeneration appears to be very reduced when chemotherapy is associated with radiotherapy.

[The hematopoietic stem cell and the stromal microenvironment]. / Les cellules souches hématopoïétiques (CSH) et microenvironnement stromal.

The hematopoietic system is the adult cellular model in which the biology of the stem cells is the best known and may be a model for numerous other tissues. This model is theoretically based on a hierarchy of cells, which begin on a stem cell that differentiates into mature cells through a large number of cellular stages including hematopoietic progenitors. Hematopoietic stem cells have three ain properties: (1) Self-renewal capacities. However, in transplantation experiments this property is limited and may be regulated by the stem cell niche. (2) Multipotentiality. Hematopoietic stem cells are capable of differentiation towards all myeloid and lymphoid lineages. However, recent experiments suggest that, like other somatic stem cells, hematopoietic stem cells are capable