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The aim of the present study was to evaluate the capacity to expand of hematopoietic stem cell (HSC) samples from eight patients with NHL, and to follow in parallel the fate of tumor cells in four of eight samples still containing bcl2/JH+ tumor cells after CD34+ or CD19-/20-/34+ cell selection. The presence of bcl2/JH+ cells was also investigated after expansion in four of eight samples, two of which were bcl2/JH at harvesting and two which were initially bcl2/JH+ but became bcl2/JH (below the level of PCR detection) after cell selection, to assess a possible reappearance of occult tumor cells after expansion culture. We used culture conditions that we previously had established to allow high level expansion of normal precursors, progenitors and LTC-ICs. In this study, particular attention was given to the role of Flt3-ligand, known to favor the growth of B cells. The expansion conditions were: 1.5 x 10(3) cells/ml in serum-free medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte-stimulating factor (G-CSF), erythropoietin (Epo) +/- Flt3-ligand (Flt3-L) for 10 days. After culture, total cells, CFU-GMs, BFU-Es and LTC-ICs were expanded to a mean of 833-, 6.6-, 4.6-, and 1.8-fold, respectively with the cocktail of cytokines not including Flt3-L. When Flt3-L was added, the mean expansion values were 1095-, 31-, 15- and three-fold, respectively. Residual bcl2/JH+ cells present in four of eight samples before expansion were not detected after expansion. Similarly, no tumor cells reappeared after expansion of the two samples which had become negative after selection, as well as in the two samples which were bcl2/JH- at harvesting. These results suggest first that ex vivo expansion of hematopoietic stem cells in patients with non-Hodgkin's lymphoma is feasible without incurring the parallel risk of amplifying tumor cells; second, that Flt3-L did not stimulate the growth of tumor cells while it clearly favored the growth of normal progenitors.

Several prospective randomized trials in acute myelocytic leukemia (AML) documented a lower relapse rate with autologous bone marrow transplantation (ABMT) than with conventional chemotherapy. However, they also identified some transplant difficulties, such as failure to collect sufficient numbers of stem cells, slow kinetics of engraftment, and a high transplant-related mortality that diminished or negated positive impact on overall survival. Data for ABMT are inconclusive in acute lymphocytic leukemia (ALL) in adults. We retrospectively analyzed patients with acute leukemia autografted with marrow purged with mafosfamide after January 1983 in our institution. The population comprised 229 consecutive patients; 165 with AML [123 in first remission (CR1), 32 in second remission (CR2)]; 61 with ALL (46 in CR1, 4 in CR2); and 3 with undifferentiated acute leukemia. All patients were autografted with marrow purged with mafosfamide. Mafosfamide was given at a constant dose of 50 μg/mL in 103 and adjusted individually to produce a CFU-GM LD 95 (5% residual CFU-GM post purging) in 126. The outcome was analyzed for correlation with patient characteristics, the disease including cytogenetics, and the graft itself. Prognostic factors identified by multivariate analysis were used to derive a prognostic classification. Patients receiving higher doses of marrow submitted to purging (>5.46 × 10 4 CFU-GM/kg) experienced a lower treatment-related mortality (RR = 0.11, p=0.005) and a higher leukemia-free (RR = 0.5, p=0.005) and overall survival (RR = 0.4, p=0.001). Patients receiving 5.46 × 10 4 CFU-GM/kg and doses actually infused post purging of ≤0.02 × 10 4 /kg had a treatment-related mortality of only 2 ± 2%, a leukemia-free survival of 70%, and an overall survival of 77 ± 7% at 10 years. In this study of autotransplantation for acute leukemia using mafosfamide-purged marrow, the stem cell dose used for purging and the intensity of purging were the most important factors predicting outcome.

Resume En trente ans, le monde hematologique est passe du concept de greffe de moelle a celui de greffe de cellules souches hematopoietiques (CSH) sanguines ou placentaires, du concept de l'allogreffe a celui de l'autogreffe, du greffon non manipule a l'hyperselection, de la therapie cellulaire hematopoietique a l'immunotherapie. Les indications de ces greffes se sont precisees dans le domaine des maladies malignes et s'etendent maintenant aux pathologies auto-immunes. Une meilleure connaissance de la CSH permet un controle de sa proliferation et de sa differenciation, ouvrant le vaste champ de l'expansion ex vivo des differents compartiments hematopoietiques. Depuis peu, de nouvelles cellules souches sont mises en evidence, montrant ainsi qu'une cellule differenciee garde ses potentialites totipotentes: une cellule nerveuse peut en effet se differencier en CSH qui peut donner ellememe naissance a l'hematopoiese, a des cellules mesenchymateuses ou a des hepatocytes. De nouveaux outils se developpent: cellules embryonnaires humaines, biomateriaux, materiaux fonctionnalises, genie tissulaire, ouvrant la voie a l'ingenierie cellulaire des annees 2000.

The aim of this study was to evaluate the ex vivo expansion of normal CD34 + cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF ± MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10 3 to 1 x 10 5 CD34 + cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39 ± 0.5% in expanded cultures derived from fractions initiated at 5 x 10 3 , 104 or 10 5 cells/ml and 45 ± 6% in cultured cells obtained from starting fractions containing 5 x 10 4 cells/ml, as compared to 58 ± 4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H 2 O 2 , although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45 ± 4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.

Autologous bone marrow transplantation (ABMT) after high-dose chemotherapy is recognized as a curative approach to treating hematologic malignancies and some invasive solid tumors. However, tumor cells present in the bone marrow at the time of harvesting are a potential cause for relapse. Ex vivo marrow purging with very high doses of cytotoxic agents has been introduced in an attempt to remove neoplastic cells contaminating the autograft. The procedure, however, has been limited by its high toxicity to normal bone marrow progenitor cells. In their purging procedures, investigators have used agents such as amifostine, originally developed to protect against the effects of radiation and chemotherapy. In this article, the appropriateness of protecting normal cells with amifostine during various purging procedures will be reviewed.

PURPOSE To analyze retrospectively survival and prognostic factors of patients with non-Hodgkin's lymphoma (NHL) autografted from 1979 to 1995 in a single institution. PATIENTS AND METHODS A total of 120 patients, 64 with aggressive and 56 with low-grade NHL, were autografted. The carmustine (BCNU), etoposide, cytarabine, and melphalan (BEAM) regimen was used in 104. The autograft was marrow in 101 patients. Marrow was purged in vitro by mafosfamide for 63 patients (adjusted dose [AD] in 32; unique dose [UD] in 31); 27 patients received a CD34+-selected graft. Following intensification, 45 patients received additional radiotherapy on previous sites of involvement. RESULTS Outcome at 5 years for patients transplanted with low-grade NHL in first complete remission (CR1), in first partial remission (PR1), and in second complete remission (CR2) or beyond showed an event-free survival (EFS) of 75% +/- 12%, 46% +/- 18%, and 57% +/- 24%, a relapse incidence (RI) of 21% +/- 12%, 49% +/- 19%, and 43% +/- 25%, and a transplant-related mortality (TRM) of 5% +/- 5%, 10% +/- 7%, and 0%, respectively. For patients with aggressive NHL transplanted in CR1, in PR1, in CR2 or beyond, and in resistant relapse or in primary refractory disease, the EFS was of 73% +/- 9%, 58% +/- 19%, 29% +/- 16%, and 10% +/- 9%, the RI 22% +/- 9%, 14% +/- 9%, 77% +/- 18%, and 66% +/- 20%, and the TRM 6% +/- 6%, 32% +/- 21%, 11% +/- 10%, and 71% +/- 22%, respectively. In patients autografted upfront in first remission, additional radiotherapy was associated with a higher EFS, in univariate (P = .03) and multivariate analysis (P = .02, relative risk [RR] = .021). The role of graft purging with mafosfamide on the outcome reflected by the dose of colony-forming unit-granulocyte-macrophage (CFU-GM) per kilogram infused postpurging was assessed by univariate analysis: patients in first remission who received lower doses of CFU-GM had a lower RI and a higher EFS. CONCLUSION This retrospective analysis suggests that marrow purging and posttransplant radiotherapy improve the outcome of patients with NHL autografted in first remission.

High dose radiation exposures involving medical treatments or accidental irradiation may lead to extended damage to the irradiated tissue. Alleviation or even eradication of irradiation induced adverse events is therefore crucial. Because developments in cell therapy have brought some hope for the treatment of tissues damages induced by irradiation, the Institute for Radiation and Nuclear Safety contributed to establish the clinical guidelines for the management of accidentally irradiated victims and to provide the best supportive care to patients all over the world. In the past 15 years, we contributed to develop and test cell therapy for protection against radiation side effects in several animal models, and we proposed mechanisms to explain the benefit brought by this new therapeutic approach. We established the proof of concept that mesenchymal stem cells (MSCs) migrate to damaged tissues in the nonobese diabetic/severe combined immunodeficiency immunotolerant mice model and in non-human primate after radiation exposure. We showed that the intravenous injection of MSCs sustains hematopoiesis after total body irradiation, improves wound healing after radiodermatitis and protects gut function from irradiation damages. Thanks to a tight collaboration with clinicians from several French hospitals, we report successful treatments of therapeutic/accidental radiation damages in several victims with MSC infusions for hematopoiesis correction, radio-induced burns, gastrointestinal disorders and protection homeostatic functions of gut management after radio-therapy.

Blood-transfusion centers regularly face the challenge of donor blood shortages, especially for rare blood groups. The possibility of producing universal red blood cells from stem cells industrially has become a possible alternative since the successful injection of blood generated in vitro into a human being in 2011. Although there remains many biological and regulatory issues concerning the efficacy and safety of this new product, the major challenge today for future clinical applications is switching from the current limited 2-dimensional production techniques to large-scale 3-dimensional bioreactors. In addition to requiring technological breakthroughs, the whole process also has to become at least five-fold more cost-efficient to match the current prices of high-quality blood products. The current review sums up the main biological advances of the past decade, outlines the key biotechnological challenges for the large-scale cost-effective production of red blood cells, proposes solutions based on strategies used in the bioindustry and presents the state-of-the-art of large-scale blood production.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

To circumvent the problems inherent in allogeneic bone marrow transplantation, allogeneic cord blood has been studied as an alternative source of hematopoietic stem cells. Preliminary results with cord blood from an HLA-matched sibling are encouraging, but the experience with this procedure is limited.1–5 Although some 200 transplantations of cord blood have been performed in children,6,7 few have been performed in adults. We describe an adult with chronic myelogenous leukemia who underwent the transplantation of cord-blood stem cells successfully. Case Report A 26-year-old woman was found to have chronic myelogenous leukemia in July 1990. All the leukemic cells had . . .

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.

Resume Le contenu en progeniteurs granulo-macrophagiques (CFU-GM) de 130 echantillons de moelle osseuse et de 105 echantillons de sang a ete etudie en parallele par 2 techniques, l'une en agar avec du milieu placentaire, l'autre en methyl-cellulose supplementee avec des facteurs de croissance recombinants (G-CSF, GM-CSF, IL3 et erythropoietine). Les echantillons de moelle ont ete testes frais (130), apres traitement par mafosfamide (31) et apres congelation-decongelation (33). Les resultats montrent que le nombre de CFU-GM detectees en methyl-cellulose est pour tous les types d'echantillons correle avec celui qui est detecte en agar (p = 0,0001) par une formule de type « log CFU-GM methyl = a log CFU-GM agar + b . Sur la base des correlations trouvees, les doses seuils/kg de CFU-GM detectees en methyl-cellulose necessaires dans notre experience pour constituer un greffon autologue de moelle osseuse et de sang ont ete recalculees a partir des doses seuils que nous avions precedemment determinees en agar (respectivement 104/kg et 5 × 104/kg) : ces doses sont respectivement 2,3 × 104/kg pour la moelle osseuse non traitee, et 2,1 × 105/kg pour les cellules sanguines.

The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34+ cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test.

International audience; Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. © 2012 Ferrata Storti Foundation.

*the first two authors are co-first authors Introduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process. Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1, transcription factors, heme biosynthesis enzymes followed the expected pattern. To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins. Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis. Disclosures No relevant conflicts of interest to declare.

IL2 infusion may benefit patients with haematological malignancies by lowering the disease burden. However, conflicting data have been reported on IL2 effects on myelopoiesis, in vitro as well as in vivo. In the present study we investigated the ability of IL2 to act on committed and primitive bone marrow progenitor cells in defined serum-free (SF) culture conditions which avoid many technical biases such as interference by exogenous stimulating or inhibiting factors. Low doses of IL2 (0.1-1000 U/ml) were studied without or in combination with recombinant IL3, GM-CSF and erythropoietin, in SF long-term marrow culture (LTMC). We report data in favour of an inhibitory activity of IL2 limited to committed progenitors and excluding more primitive haemopoietic stem cells, as shown by an alteration of CFU-GM proliferation during the first 5 weeks of LTMC, decreasing with time, unaffected BFU-E and increased nucleated cell production. Beyond week 5, no difference was observed between IL2 supplemented cultures and the SF control cultures. In parallel, IL2 induced the adherence of fibroblastic cells and their progeny. In addition to the inhibitory effect, IL2 appeared to limit the stimulating effect on granulopoiesis and erythropoiesis of myeloid growth factors (GF) such as combination of IL3, GM-CSF and EPO. Indeed, in SF-LTMC conditions, IL2 inhibitory effect is effective on CFU-GM production throughout the 7 weeks of LTMC and on BFU-E during the first 2 weeks only. These data confirm the interaction of IL2 with other GFs in the complex interplay of the cytokine network.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBCs generated under good manufacturing practice conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro–generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

In the context of chronic blood supply difficulties, generating cultured red blood cells (cRBCs) in vitro after amplification of stem cells makes sense. This review will focus on the recent findings about the generation of erythroid cells from induced pluripotent stem (iPS) cells and deals with the hurdles and next developments that will occur.The most proliferative source of stem cells for generating cRBCs is the cord blood, but this source is limited in terms of hematopoietic stem cells and dependent on donations. Pluripotent stem cells are thus the best candidates and potential sources of cRBCs. Critical advances have led towards the in-vitro production of functional RBCs from iPS cells in the last few years.Because iPS cells can proliferate indefinitely and can be selected for a phenotype of interest, they are potential candidates to organize complementary sources of RBCs for transfusion. Proof of concept of generating cRBCs from iPS cells has been performed, but the procedures need to be optimized to lead to clinical application in blood transfusion. Several crucial points remain to be resolved. Notably these include the choice of the initial cell type to generate iPS cells, the method of reprogramming, that is, to ensure the safety of iPS cells as clinical grade, the optimization of erythrocyte differentiation, and the definition of good manufacturing practice (GMP) conditions for industrial production.

Background This study is the first step toward use of mesenchymal stem cells (MSCs) in therapy to alleviate the effects of radiation exposure in patients. MSCs improve repair in various organs and tissues, in particular after irradiation. However, before using MSCs in a cancer environment, for instance after radiotherapy, it must be established that MSC injections do not promote malignant cells proliferation. Methods A rat model of colorectal carcinogenesis close to human cancer was used as preclinical model. The carcinogen N-methyl-N ′ -nitro-N-nitroso-guanidine (MNNG) was instilled in rats by four intrarectal deposits within 2 weeks, and two MSC injections (10 7 cells per rat) 2 and 4 weeks after the end of carcinogen treatment. Tumours were then examined 32 and 52 weeks after initiating MNNG treatment. Another cohort of rats entered a survival experiment. Findings MSC treatment reduced cancer significantly, lowering the number of adenomas and adenocarcinomas and extending the lifespan of rats. The anti-cancer effect of MSCs was mediated by their immunological properties. In adenocarcinoma of MSC-treated rats, CD68+ monocyte/macrophage infiltration was lowered and CD3+ lymphocytes increased. MSCs induce macrophages to turn into regulatory cells involved in phagocytosis, inhibiting the production of proinflammatory cytokines. MSCs decrease NK cells and rTh17 cell activities, Treg recruitment, CD8+ lymphocytes, and endothelial cell number and restore Th17 cell activity. MiRNA mi-150 and miRNA-7 are the key effectors. Mi-150 inhibits tumour invasion and mi-RNA-7 negatively regulates the EGFR/AKT pathway, promoting cell death. Interpretation MSC infusion has a durable action on colon cancer development by modulating the immune component of the tumour microenvironment. For the first time, two mi-RNA were identified as responsible for the anti-cancer effect of MSCs. We propose that in this model of colorectal cancer early bone-marrow-derived MSC injections, by a long-term effect, reoriented tumour immune response and induced regression of colonic carcinogenesis.

This article describes the research in progress that will permit the large-scale production of human red blood cells from hematopoietic stem cells. It also discusses the current state of this research, suggests the obstacles to be overcome to pass from the laboratory model to clinical practice, and analyzes the possible indications in the medium and long term. The potential interest of pluripotent stem cells as an unlimited source of red blood cells is considered. If it succeeds, this new approach could mark a considerable advance in the field of transfusion.

The work on human embryonic stem cells is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardisation between projects and publications very difficult. Clearly the consequence of using such cells would be wasted time and resources but, more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. This guidance document represents the outcome of the first meeting of the group named The International Stem Cell Banking Initiative held in October 2007. The document has been prepared from the perspective of hESC culture but, in many respects, is broadly applicable to all human stem cell lines including induced pluripotent stem cell lines.

International audience; Nasal-type natural killer (NK) cell lymphoma is an infrequent aggressive malignant disease with very poor prognosis. We aimed to explore the possible role of the transcription factor STAT3 in the pathophysiology of this malignancy, as it was involved in oncogenesis and chemoresistance. For this, we established and characterized a continuous interleukin 2-dependent NK cell line (MEC04) from a patient with a fatal nasal-type NK-cell lymphoma. Cells harbored poor cytotoxic activity against K562 cells, and spontaneously secreted interferon-gamma, interleukin-10 and vascular-endothelium growth factor in vitro. STAT3 was phosphorylated in Y705 dimerization residue in MEC04 cells and restricted to the nucleus. Y705 STAT3 phosphorylation involved JAK2, as exposure of cells to AG490 inhibitor inhibited Y705 STAT3 phosphorylation. By using recombinant transducible TAT-STAT3-beta (beta isoform), TAT-STAT3Y705F (a STAT3 protein mutated on Y705 residue, which prevents STAT3 dimerization) and peptides inhibiting specifically STAT3 dimerization, we inhibited STAT3 phosphorylation and cell growth, with cell death induction. Finally, STAT3 was phosphorylated in Y705 residue in the nuclei of lymphoma cells in eight/nine patients with nasal-type NK/T-cell lymphoma and in YT, another NK cell line. Our results suggest that STAT3 protein has a major role in the oncogenic process of nasal-type NK-cell lymphomas, and may represent a promising therapeutical target.

We have sufficient knowledge of the biology of hematopoietic stem cells to hope that we might generate human red blood cells in the laboratory. It may soon be possible to produce enough to transfuse "cultured" red blood cells to manufacture human red blood cells from hematopoietic stem cells for transfusion purposes. This article describes progress and the challenges that remain in the search for in vitro generated red blood cells that can be efficiently manufactured in high volumes and given to any recipient.

We describe a technological approach permitting the massive expansion of CD34(+) stem cells and their 100% conversion ex vivo into mature red blood cells (RBC). The protocol comprises three steps: a first step consisting of cell proliferation and induction of erythroid differentiation in a liquid medium without serum in the presence of growth factors (GF), a second based on a model reconstitution of the medullar microenvironment (ME) (human MSC or murine stromal cells) in the presence of GF, and a third in the presence of the ME alone, without any GF. This work highlights the impact of the ex vivo microenvironment on the terminal maturation of erythroid cells. A critical point is that the RBC generated in vitro have all the characteristics of functional native adult RBC. Moreover, this new concept of 'cultured RBC' (cRBC) is important for basic research into terminal erythropoiesis and has major clinical implications, especially in transfusion medicine. The three-step protocol can be adapted to use hematopoietic stem cells (HSC) from diverse sources: peripheral blood, bone marrow or cord blood.

International audience; BACKGROUND: The prognostic value of the ectopic activation of TLX3 gene expression, a major oncogenetic event associated with pediatric T-cell acute lymphoblastic leukemia, is controversial. Likewise, the frequency and the prognostic significance in pediatric T-cell acute lymphoblastic leukemia of the newly characterized NUP214-ABL1 fusion transcript is not yet clear. DESIGN AND METHODS: Two hundred children with T-cell acute lymphoblastic leukemia were treated in the French FRALLE-93 study from 1993 to 1999. The expression of TLX3, TLX1 and SILTAL1 genes was analyzed in samples from 92 patients by real-time quantitative reverse transcriptase polymerase chain reaction. Most of these samples were further studied for NUP214-ABL1 and CALM-AF10 fusion transcripts. RESULTS: The median follow-up was 7.9 years. At 5 years the overall survival (+/- standard deviation, %) was 62 (+/-3%) and leukemia-free survival was 58 (+/-3%). Patients with T-cell acute lymphoblastic leukemia positive for TLX3 had a poorer survival compared to those with T-ALL negative for TLX3 (overall survival: 45+/-11% vs. 57+/-5%, p=0.049). In multivariate analysis, TLX3 expression was an independent adverse risk factor predicting relapse with a hazard ratio of 2.44 (p=0.017) and an overall survival with a hazard ratio of 3.7 (p=0.001). NUP214-ABL1 was expressed in 16.6% of patients with TLX3-positive T-ALL (3 of 18); all of the patients with this association died before completion of the treatment. SILTAL expression did not significantly affect the prognosis of patients with T-cell acute lymphoblastic leukemia. Only three of 92 patients expressed the TLX1 gene and all three are alive. CONCLUSIONS: TLX3 gene expression is an independent risk factor predicting poor survival in childhood T-cell acute lymphoblastic leukemia. When co-expressed with TLX3, NUP214-ABL1 transcripts may increase the risk of poor survival.

For several years, researchers have been trying to find a substitute for red blood cells (RBC). The development of chemical or natural molecules to replace hemoglobin has nevertheless proved difficul

There is difficulty in obtaining adequate supplies of blood components, as well as disappointing performance of stabilized or recombinant hemoglobins, limited indications of oxygen transporters (perfluorocarbons), and slow development of "universal" red blood cells (RBCs). There is, therefore, a need for complementary sources of RBCs for transfusion. Thus, an attempt to generate erythroid cells in vitro makes good sense. We describe in this article a methodology permitting the massive ex vivo production of mature human RBCs having all the characteristics of native adult RBCs from hematopoietic stem cells of diverse origins: blood, bone marrow, or cord blood. This protocol allows both the massive expansion of hematopoietic stem cells/progenitors and their complete differentiation to the stage of perfectly functional mature RBCs. The levels of amplification obtained (10(5) to 2 x 10(6)) are compatible with an eventual transfusion application. We discuss in this article the state of the art of this new concept and evoke possible obstacles that need to be overcome to pass from a laboratory model to clinical practice. We analyze its possible indications in the medium and long term, discuss the economic aspects, and raise the question: Can we afford the luxury of developing this approach, one that could represent a considerable advance in blood transfusion?

We describe a technical approach permitting massive expansion of CD34+ stem cells (up to 1.95 x 10(6)-fold) and their full ex vivo conversion into mature red blood cells (RBCs). This three-step protocol can be adapted to hematopoietic stem cells (HSC) of various origins. First, cell proliferation and erythroid differentiation are induced in serum-free media supplemented with stem cell factor, interleukin-3 and erythropoietin (Epo) for 8 days. The cells are then co-cultured with either the murine stromal cell line MS-5 or human mesenchymal cells for 3 days in the presence of Epo alone. Finally, all exogenous factors are withdrawn and the cells are incubated on a simple stroma for up to 10 days. The ex vivo microenvironment strongly influences both the terminal maturation of erythroid cells and hemoglobin (Hb) synthesis. Critically, in vitro-generated RBCs have all the characteristics of functional native adult RBCs in terms of their enzyme content, membrane deformability, and capacity to fix and release oxygen. In addition, their behavior in the murine NOD/SCID model mirrors that of native RBCs. This new concept of "cultured RBCs" (cRBC) has major implications for basic research on terminal erythropoiesis and for patient management. Currently, the potential yield of functional red cells is compatible with clinical requirements, as several units of packed RBCs can be produced from a single donation. Importantly, infused cRBC would all have a life-span of about 120 days, whereas the mean half-life of normal donor RBCs is only 28 days. This would help to minimize the transfusion exposure of patients requiring regular treatment, thereby reducing the risk of iron overload and allo-immunization. The use of autologous CD34+ cells isolated from leukapheresis samples could be beneficial for patients who no longer tolerate allogeneic RBCs. This new method should also prove useful for analyzing the mechanisms of terminal erythropoiesis, including hemoglobin synthesis. Finally, it could provide a tool for investigating the lifecycle of blood parasites such as Plasmodium, the agent of malaria.

Introduction Acute myeloid leukemia (AML) is an aggressive malignancy caused by the accumulation of multiple oncogenetic mutations occurring in a single lineage of hematopoietic progenitors. AML is rare in children and the mutations found are partially different from those in adults, and for some with a lower frequency. Thus, clonal evolution leading to pediatric AML may be specific, and has not been described yet. Methods To define clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trio of specimens (diagnosis, germline and relapse) in a 9-years old girl presenting AML FAB M5a with t(9;11)(p22;q23) MLL-AF9 and trisomy 8. At diagnosis, we focused on 3 non-silent somatic mutations candidate for leukemogenesis process, confirmed by Sanger method: EED (R355*), GSDMC (R40*) and ELK1 (3’ UTR). In the same time, we performed cell cultures from bone marrow mononucleated cells at diagnosis. CD34 and CD38 cells were cultured either in liquid long term culture medium (LTC IC) or methylcellulose medium. Results: A total of 512 colonies were collecte. Our 3 interest mutations and trisomy 8 were tracked by allele-specific PCR, and MLL rearrangement detected by FISH, individually in 267 from the 512 colonies. Exploitable results were found in 164 colonies. Through these results in the different cell populations, we were able to establish the clonal architecture at diagnosis. MLL-AF9 fusion and EED mutation were found together as the first concomitant occurring events in the leukemic clone. Then genotyping of the colonies demonstrated that ELK1 mutation, GSDMC mutation, and trisomy 8 were successively acquired. Additional later mutations such as ASXL1 (frameshift), PTPN11 (E76K), EMP2 (3’UTR) and DGCR14 (P314S) were detected in the relapse sample. Discussion The 3 mutations studied in the colonies may impact the progression of the leukemic clone by dysregulating several cellular pathways and networks. First, EED is an essential non-catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates gene silencing through catalysis of histone H3K27 methylation. PRC2 is known to be enhanced in solid neoplasms such as prostate cancer. On the contrary, in myeloid malignancies and myelodysplasic syndromes, it has been recently demonstrated that mutations involving PRC2 subunits (EED, SUZ12 and EZH1/2) were hypomorphic. These loss-of-functions mutations were responsible for chromatin relaxation and induced transcription of genes promoting self-renewal such as HOXA9. Nevertheless, recent sh-RNA studies in a murine model of MLL-AF9 leukemia demonstrated that residual PRC2 enzymatic activity after EED mutation is needed to unable leukemia growth. These data are coherent with our finding that EED mutation is an early event in leukemogenesis, in cooperation with MLL-AF9 rearrangement. Secondly, ELK1 is targeted by RAS-MAPK pathway, thus its mutation can confer an increased proliferation potential when acquired by the leukemic clone, after its maturation has been blocked and its self-renewal increased through previous MLL rearrangement and EED mutation. Finally, GSDMC may be implicated in monocyte count regulation, and mutated in other neoplasms such as melanoma. As a consequence, it is likely that its mutation occurs lately in the evolution of the monoblastic leukemic clone of our patient. The latest event in the clonal evolution in our patient at diagnosis is the acquisition of trisomy 8. Conclusion This study highlights the clonal evolution in one pediatric AML, and paves the way for further studies to better understand clonal evolution in children. Elucidating, the succession and the cooperation between driver and secondary mutations, is important for both understanding leukemogenesis and developing innovative therapeutic agents targeting founding anomalies in the leukemic clone at its most precocious stage. Moreover, discovering clonal architecture also unable to find new minimal residual disease markers to assess the therapeutic response and risk stratification. Disclosures No relevant conflicts of interest to declare.

THE MAIN DISADVANTAGES of transplantation of cord blood (CB) hematopoietic stem or progenitor cells (HSPC) are the delayed neutrophil and platelet reconstitution coupled with the difficulty in obtaining a large enough graft for patients weighing more than 30 kg (1). Both problems could be overcome by ex vivo expansion of CB progenitors. In most CB banks, the whole CB samples are stored in individual or double bags. The ex vivo expansion of progenitors in whole blood samples is inefficient; thus, a CD341 selection before an expansion procedure is a prerequisite. However, this selection using frozen-thawed blood samples is impaired by aggregate formation (“clumping”). To overcome this problem, addition of recombinant human deoxyribonuclease (DNase) has been proposed during thawing of CB cells prior to transplantation (2) or expansion (3). We have extended this procedure to clinical-grade conditions incorporating subsequent CD341 cell selection and expansion in two-step culture conditions. Two bags [(Macopharma, Lille, France) GSR 7000 A), each containing 125 ml of CB and 125 ml of cryoprotecting solution 20% dimethylsulfoxide (DMSO), from a single CB and frozen by using a controlled-rate procedure and stored in liquid nitrogen, were thawed in a water bath at 37°C. DNase (Pulmozyme Roche, Neuilly, France) (3750 units) and the MgCl2 solution (Cooper, Mellin, France) (0.5 M, 250 ml) were added to each bag. Two samples were pooled and then rehydrated slowly (10 min at 20–22°C) by adding 250 ml of Dextran 40-sorbitol solution (Braun Medical, Boulogne, France) and 3.3 ml of Gamma IV (0.5 g LFB). The cell suspension was filtered, incubated with anti-CD34, and transferred into the separation chamber (system Isolex 300i, Baxter, Deerfield, IL). An additional 7500 Units of DNase and 500 ml of MgCl2 were added before the addition of immunomagnetic microbeads (Baxter kit). The suspension was processed on Isolex 300i. The CD341 cells were transferred into 50-ml conical tubes and centrifuged for 10 min at 1500 rpm. The supernatant was removed, and the cells were resuspended in 10 ml of serum-free culture medium (Irvine Scientific, Santa Ana, CA). The CD341 cells were expanded in the same serum-free medium (200 ml; 10,000 cells/ml) in gas-permeable bags (Opticyte 390 cm2, Baxter, Maurepas, France) in the presence of stem cell factor (SCF; Amgen, Thousand Oaks, CA), megakaryocyte growth and development factor (MGDF) (Amgen), Flt3 ligand (RD Median 3.333 106 n 5 5) CD341 cells per sample (Fig. 1A), i.e., a mean recovery of 48.5% with respect to the values found in fresh CB samples (Fig. 1B). The purity of these

Peripheral blood CD34+ cell samples from eight children with advanced neuroblastoma and from 10 healthy adult donors were seeded at 5 x 10(4) cells/ml in stroma-free, serum-free medium with FL, SCF, MGDF (100 ng/ml each), G-CSF, IL6 (10 ng/ml each) and IL3 (5 ng/ml), and incubated for 10 days. The levels of expansion of PBCD34+ cells observed in neuroblastoma patients, with up to 214-fold expansion for total nucleated cells, 39-fold for CD34+ cells, 79-fold for CFU-GM and nine-fold for LTC-IC were identical to those obtained with PBCD34+ cells of healthy donors (P/=0.5). All samples from patients with neuroblastoma and five donor's PBCD34+ cell samples contaminated with IMR-32 neuroblasts, were screened for the number of tyrosine hydroxylase (TH) mRNA transcript using LightCycler software. In all samples, progressive 1.9-4.4 log decreases in the number of TH transcripts were observed between days 0 and 10 of expansion. Our results show that in extensively pretreated children with neuroblastoma, the culture conditions that were effective for BM and CB cell expansion can generate an expansion of PBCD34+ cells and provide a purge of tumour cells.

In vitro production of red blood cells (RBCs) from stem cells (SCs) for transfusion is emblematic of what regenerative medicine could be in the near future [1]. This project of proposing an alternative to conventional transfusion, includes four challenges – scientific, technological, public health and social – that must be overcome to attain large-scale clinical development. First, a scientific challenge: recreate in vitro a fundamental physiological mechanism – the complete and terminal maturation of RBCs from a SC population. The goal is to reach the ultimate stage of the erythroid lineage, cells that specialize in oxygen transport to the point of losing all other functions, even eliminating their nucleus. The complexity of this concept resides in that we need to identify the best source of SCs to attain a double goal: qualitative (mature and functional cells) and quantitative (massive amplification allowing transfusion applications). When this project was initiated in early 2000, the target population was limited to hematopoietic SCs from bone marrow, blood or umbilical cord. Today, other major SCs are putative candidates, the pluripotent, embryonic SCs or adults SCs (induced pluripotent SCs; iPSCs) [2]. Immortalized lines of CD34 cells will probably soon be available also. Indeed, while hematopoietic SCs theoretically allow our quantitative goal of RBC production for transfusion to be reached, their availability is dependent on donors and is thus limited. These are the limitations. Because iPSCs can proliferate indefinitely, they are potential candidates to provide complementary sources of RBCs for transfusion. Proof of concept for generating human RBCs from SCs has been performed, but the procedures need to be optimized to lead to clinical application in blood transfusion [3]. Several crucial points remain to be resolved. Notably, these include: the choice of the initial cell type to generate iPSCs; the method of reprogramming to ensure their safety as clinical-grade cells; the optimization of erythrocyte differentiation; and the definition of GMP conditions for industrial production. Thus, while we know the goal to attain, we do not know yet from where to start. It is a paradox! Fundamental science moves faster than industrial development. This problem is common to all fields of regenerative medicine. Let us take the example of cardiac regeneration. Who can say today which is the appropriate target: total bone marrow cells, hematopoietic CD34 cells, CD133 cells, mesenchymal SCs, endothelial cells, embryonic SCs or iPSCs? All leads must be investigated without exclusion or bias.

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.

The ex vivo expansion of hematopoietic stem cells (HSC) for clinical use is now recognized to be a feasible and very promising approach for hematotherapy. Expansion of specific HSC subsets is required for different clinical applications, for example, to increase the number of mature cells, to produce specific cells for adoptive therapy, or to increase the number of primitive stem cells available for engraftment. Although hematopoietic growth factors can play an important role in this setting, in this review we emphasize that other variables affect the outcome of stem and progenitor cell expansion. These variables include the serum supplement, the purity of CD34(+) cells, the initial cell concentration, and the duration of culture. It is also essential to define standard culture conditions for normal stem cells and to limit or prevent expansion of residual tumor cells. In clinical applications, determination of the hematopoietic value of the expanded population is mandatory. Thus, we have to demonstrate the expansion of primitive hematopoietic progenitor and stem cells, with maintenance of their hematopoietic potential as assessed by in vitro or in vivo assays. We draw attention to the challenges in the clinical application of ex vivo expansion. These include the establishment of well-defined experimental conditions and the determination of the hematopoietic value of the expanded grafts, whatever the graft source: bone marrow, mobilized peripheral blood, or cord blood. Future studies hopefully will optimize these procedures and allow not only expansion but engineering of defined cellular functions as HSCs grow under defined conditions.

We tested two positive selection techniques for separation of CD34+ cells from bone marrow and analyzed the yields of CD34+ cells, BFU-E, CFU-GM, CFU-MK and LTC-IC after selection and expansion. An immunoadsorption procedure (CellPro) and an immunomagnetic (Baxter) CD34+ cell separation method were employed to purify the same bone marrow samples from seven normal subjects. Mean yields of CFU-GM and CFU-MK and absolute numbers of LTC-ICs were not different in the two purified cell populations. In contrast, the mean recovery of BFU-E was significantly lower for the immunoadsorption (21 +/- 14%) than for the immunomagnetic technique (44 +/- 27%). After separation, CD34+ cells were evaluated in 10-day liquid cultures for their expansion capacity in terms of total cells and progenitors. The expansion capacity of progenitors such as CFU-GM, CFU-MK and especially BFU-E selected by immunoadsorption was higher than the capacity of progenitors obtained by immunomagnetism, although final total and progenitor cell numbers are similar. Our results suggest that the populations separated by the two techniques differ mainly in the expansion capacity of progenitors and in the recovery of BFU-E after the selection procedure. These differences between two methods, which already are widely employed in research and in clinical transplantation, should be taken into account when considering the aims of the experiments.

The aim of the study is to define the ability of Flt3 ligand, MGDF, Epo and G-CSF to modulate the expansion of different hematopoietic compartments in association with a basic cocktail of SCF + IL-3 + IL-6 (S36). CD34+ cells from normal bone marrow were cultured in stroma-free, serum-free medium for 10 days. Using various concentrations of cytokines, total cells could be expanded up to 5200-fold, CD34+ cells up to 78-fold, CFU-GM up to 143-fold, BFU-E up to 46-fold, CFU-MK up to six-fold and LTC-IC up to four-fold. The results were assessed by multiparametric analysis of variance. Three factors had a significant stimulatory effect on the late precursor compartment: Epo (P < 10(-5)), G-CSF (P=5 x 10(-3)) and FL (P=10(-5)). Two were critical for CD34+ cell expansion: FL (P=4 x 10(-5)) and Epo (P=6 x 10(-5)), while two were critical for BFU-E expansion: MGDF (P=8 x 10(-4)) and FL (P=0.017). FL strongly stimulated CFU-GM expansion (P < 10(-5)), whereas none of the growth factors studied had any effect on CFU-MK. FL (P=10(-4)) and MGDF (P=0.002) were essential to obtain high levels of expansion of LTC-IC as determined in limiting dilution assays. In the light of the above results showing a preferential effect on the expansion of precursor cells (3080-fold), CD34+ cells (53-fold), CFU-GM (134-fold), BFU-E (46-fold) and LTC-IC (five-fold), the combination SCF, IL-3, IL-6, FL, MGDF, Epo and G-CSF was chosen as a putative cytokine cocktail for further studies on long-term culture. Sustained production of precursor cells, progenitor cells, LTC-IC and E-LTC-IC for up to 100 days reflects the persistence of very primitive stem cells. This suggests that these populations are probably able to undergo self-renewal divisions. The above combination of cytokines meets the required criterion for potential clinical application, which may be defined as an effective capacity to expand all cell compartments, using as the starting material high concentrations of low purity CD34+ cells.

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Abstract SCI-39 The generation of red blood cells (RBCs) in vitro using biotechnologies could represent an interesting alternative to classical transfusion products, in that it would combine adequate supplies with the specific production of blood products of a particular phenotype and the reduction of infection risks. This presentation will review how it is now possible to obtain in vitro complete maturation of the erythroid line to the stage of enucleation, starting from hematopoietic stem cells (HSCs) from peripheral blood, bone marrow or umbilical cord blood, or embryonic stem cells or adult pluripotent stem cells (induced pluripotent stem cells, iPSCs). This presentation will discuss how the functionality of cultured human RBCs (cRBCs) is settled in terms of deformability, hemoglobin maturation, oxygen carrying capacity, enzyme content, and terminal maturation from the reticulocyte stage to mature RBC after infusion into the NOD/SCID mouse model. The clinical feasibility of this concept has recently been demonstrated by reporting that cRBCs generated in vitro from peripheral HSCs under GMP conditions encounter in vivo the conditions required for their maturation and that they persist in the circulation for several weeks in humans. These data have established the proof of principle for transfusion of in vitro-generated RBCs and the pathway toward new developments in transfusion medicine. The most proliferative source of stem cells for generating cRBCs is cord blood, but it is limited in terms of HSCs and is dependent on donations. Pluripotent stem cell technology represents a potentially unlimited source of RBCs and opens the door to the development of a new generation of allogeneic transfusion products. Because iPSCs can be selected for a phenotype of interest, they are obviously the best candidate for organizing complementary sources of RBCs for transfusion. It is established that only three human iPSC clones would have been sufficient to match more than 99 percent of the patients in need of RBC transfusions. As a whole, a very limited number of RBC clones would provide for the needs of most alloimmunized patients and those with a rare blood group. Generating cRBCs from iPSCs has been done but needs to be optimized to lead to a clinical application in blood transfusion. Several crucial points remain to be resolved, notably, the choice of the initial cell type, the method of reprogramming (i.e., to ensure the safety of the iPSCs and to ensure their clinical grade), the optimization of the erythrocyte differentiation, and the definition of GMP conditions for industrial production. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, this presentation will highlight the potential applications for alloimmunized patients and those with a rare blood group. Disclosures: No relevant conflicts of interest to declare.

We compared the effects of amifostine (WR-2721) on the cytotoxicity of mafosfamide or 4-hydroperoxycyclophosphamide (4-HC) in normal marrow progenitor cells (CFU-GM) and leukaemic progenitor cells (CFU-L) during ex vivo purging for autologous bone marrow transplantation (ABMT). Mononuclear cells (MNC) were incubated with amifostine 3 mg/ml for 15 min, washed, and subsequently tested for their sensitivity to mafosfamide or 4-HC (20-200 micrograms/ 10(7) MNC/ml). The LD95 was significantly higher among amifostine-treated cells for PCM-CFU-GM in 6 of 13 patients and for 5R-CFU-GM in 4 of 10 patients (P0.05). In contrast, amifostine exhibited no protective effects upon CFU-L. The results of this study will show that amifostine protects normal late and early progenitor cells for the toxic effects of cyclophosphamide derivatives while preserving their antileukaemic effects. These results suggest that amifostine has therapeutic value as a protective agent for normal marrow progenitor cells during ex-vivo purging of bone marrow for ABMT.

A total of 125 acute leukemia adult patients were autografted with bone marrow (BM) purged by mafosfamide (ASTA Z) during the period of January 1983 to January 1993. The median follow-up period was 64 months (range, 3 to 126). There were 84 acute myeloblastic leukemias (AMLs) and 41 acute lymphoblastic leukemias (ALLs). At time of autologous BM transplantation (ABMT); 64 AMLs were in first complete remission (CR1), and 20 were in second CR (CR2); 35 ALL were in CR1, and 6 were in CR2. The median age of the patients was 33 years (range, 16 to 55). The median interval between achieving CR and autografting was 5 months (range, 1.3 to 23). The pretransplant regimen consisted of cyclophosphamide (120 mg/kg) and total body irradiation. All patients were grafted with autologous BM treated in vitro with mafosfamide used at levels individually adjusted in 95 patients and at a standard dose in 30 patients. The initial richness in granulomacrophagic progenitors (CFU-GM) of the harvested BMs was 5.16 x 10(4) CFU-GM/kg (range, 0.55 to 33). After mafosfamide purging, the residual CFU-GM number was 0.021 x 10(4)/kg (range, 0 to 1.78). The probability of successful engraftment was significantly higher and the time to engraftment was significantly shorter in ALL. Of 33 patients grafted with BM containing no residual CFU-GM, those with AML (n = 22) had platelet recoveries that were significantly longer than those for AML patients receiving BM with residual CFU-GM. At 8 years, patients autografted in CR1 for AML and ALL had a leukemia-free survival (LFS) of 58% and 56%, respectively, with a relapse incidence (RI) of 25% and 37%, respectively. Patients autografted in CR2 for AML had an LFS of 34% and an RI of 48% at 5 years. The incidence of late relapses was significantly higher in ALLs. By multivariate analysis, four factors were found to influence favorably engraftment in addition to a diagnosis of ALL, a younger age, ABMT performed in CR1, the adjusted dose technique of purging, and a shorter interval from CR to ABMT. Two factors were correlated with a better outcome. (1) The LFS was significantly higher and the transplant-related mortality significantly lower in patients who received richer BM. (2) The RI was significantly lower in patients autografted within 150 days from CR. Our results reinforce the view that ABMT is one approach to improve the outcome of adult patients with acute leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Five patients with resistant non-Hodgkin lymphoma (NHL) were given granulocyte-macrophage colony-stimulating factor (GM-CSF, 250 micrograms/m2 daily) after the BEAM pretransplant chemotherapy regimen (carmustine 300 mg/m2, etoposide 1.2 g/m2, cytarabine 800 mg/m2, melphalan 140 mg/m2) because persistent lymphoma cell infiltration of the bone marrow precluded autologous bone-marrow transplantation (BMT). In three patients full haemopoietic reconstitution occurred, with similar kinetics to that seen after autologous BMT. The other two patients died without sustained haemopoietic recovery. GM-CSF may replace autologous BMT in highly selected cases of NHL with progressive disease and bone-marrow involvement.