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SUMMARY Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis., In Brief Gautier et al. use quantitative mass spectrometry to determine the absolute proteome composition of human erythroid progenitors throughout the differentiation process and the quantitative distribution of proteins between reticulocytes and pyrenocytes after enucleation.

Clonal history of a cord blood donor cell leukemia with prenatal somatic JAK2 V617F mutation

We here determine the influence of mesenchymal stem cell (MSC) therapy on the progression of solid tumors. The influence of MSCs was investigated in human colorectal cancer cells as well as in an immunocompetent rat model of colorectal carcinogenesis representative of the human pathology. Treatment with bone marrow (BM)‐derived MSCs significantly reduced both cancer initiation and cancer progression by increasing the number of tumor‐free animals as well as decreasing the number and the size of the tumors by half, thereby extending their lifespan. The attenuation of cancer progression was mediated by the capacity of the MSCs to modulate the immune component. Specifically, in the adenocarcinomas (ADKs) of MSC‐treated rats, the infiltration of CD68+ monocytes/macrophages was 50% less while the presence of CD3+ lymphocytes increased almost twofold. The MSCs reprogrammed the macrophages to become regulatory cells involved in phagocytosis thereby inhibiting the production of proinflammatory cytokines. Furthermore, the MSCs decreased NK (Natural Killer) and rTh17 cell activities, Treg recruitment, the presence of CD8+ lymphocytes and endothelial cells while restoring Th17 cell activity. The expression of miR‐150 and miR‐7 increased up to fivefold indicating a likely role for these miRNAs in the modulation of tumor growth. Importantly, MSC administration limited the damage of healthy tissues and attenuated tumor growth following radiotherapy. Taken together, we here show that that MSCs have durable action on colon cancer development by modulating the immune component of the tumor microenvironment. In addition, we identify two miRNAs associated with the capacity of MSCs to attenuate cancer growth. stem cells translational medicine 2019;8:285&300

We previously demonstrated that intracardiac delivery of autologous peripheral blood‐derived CD34+ stem cells (SCs), mobilized by granulocyte‐colony stimulating factor (G‐CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+‐SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G‐CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+‐cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. stem cells translational medicine 2019;8:822&832

Introductory paragraphThe successful production of Hematopoietic Stem and Progenitor Cells (HSPCs) from human pluripotent sources is conditioned by transgene delivery1-5. We describe here a dedicated and tractable one step, GMP-grade, transgene-free and stroma-free protocol to produce HSPCs from human induced pluripotent stem cells (hiPSCs). This procedure, applied to several sources of hiPSCs with equal efficiency, is based on a directed differentiation with morphogens and cytokines to generate a cell population close to nascent HSPCs or their immediate forerunners i.e., hemogenic endothelial cells6-9. Following engraftment into immunocompromised recipients, this cell population was proved capable of a robust myeloid, lymphoid and definitive red blood cell production in sequential recipients for at least 40 weeks. Further identification of the repopulating cells show that they express the G protein–coupled receptor APELIN (APLNR) and the homing receptor CXCR4. This demonstrates that the generation of bona fide HSPCs from hiPSCs without transgenes is possible and passes through an early endo-hematopoietic intermediate. This work opens the way to the generation of clinical grade HSPCs for the treatment of hematological diseases and holds promise for the analysis of HSPC development in the human species.

Extranodal, nasal-type natural killer (NK)/T-cell lymphoma (NKCL) is an aggressive malignancy with poor prognosis in which, usually, signal transducer and activator of transcription 3 (STAT3) is constitutively activated and oncogenic. Here, we demonstrate that STAT3 activation mostly results from constitutive Janus kinase (JAK)3 phosphorylation on tyrosine 980, as observed in three of the four tested NKCL cell lines and in 20 of the 23 NKCL tumor samples under study. In one of the cell lines and in 4 of 19 (21%) NKCL primary tumor samples, constitutive JAK3 activation was related to an acquired mutation (A573V or V722I) in the JAK3 pseudokinase domain. We then show that constitutive activation of the JAK3/STAT3 pathway has a major role in NKCL cell growth and survival and in the invasive phenotype. Indeed, NKCL cell growth was slowed down in vitro by targeting JAK3 with chemical inhibitors or small-interfering RNAs. In a human NKCL xenograft mouse model, tumor growth was significantly delayed by the JAK3 inhibitor CP-690550. Altogether, the constitutive activation of JAK3, which can result from JAK3-activating mutations, is a frequent feature of NKCL that deserves to be tested as a therapeutic target.

In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner., Pre-leukaemic clones, together with the propensity to cause disease in mice, are characterized by appearing early in myeloid leukaemia and being found at relapse. Here, the authors identify clones in human samples and find that they are characterized by hierarchically organized genetic lesions, which can be used to track evolution of the disease.

The increasing need for red blood cells (RBCs) together with the lack of donors have made the in vitro production of RBCs a major medical challenge.[1][1] We have recently developed a method to produce mature RBCs in vitro , starting from bone marrow, peripheral blood, leukapheresis or cord blood

Chronic skin ulcers and burns require advanced treatments. Mesenchymal Stromal Cells (MSCs) are effective in treating these pathologies. Bone Morphogenic Protein-2 (BMP-2) is known to enhance angiogenesis. We investigated whether recombinant human hBMP-2 potentiates the effect of MSCs on wound healing. Severe ulceration was induced in rats by irradiation and treated by co-infusion of MSCs with hBMP-2 into the ulcerated area which accelerated wound healing. Potentiation of the effect of MSCs by hBMP-2 on endothelial repair improved skin healing. HBMP-2 and MSCs synergistically, in a supra additive or enhanced manner, renewed tissue structures, resulting in normalization of the epidermis, hair follicles, sebaceous glands, collagen fibre density, and blood vessels. Co-localization of MSCs with CD31 + cells suggests recruitment of endothelial cells at the site of injection. HBMP-2 and MSCs enhanced angiogenesis and induced micro-vessel formation in the dermis where hair follicles were regenerated. HBMP-2 acts by causing hypoxia-inducible factor-1 α (HIF-1α) expression which impacts endothelial tube formation and skin repair. This effect is abolished by siRNA. These results propose that new strategies adding cytokines to MSCs should be evaluated for treating radiation-induced dermatitis, burns, and chronic ulcers in humans.

Extensive mutational status of genes and clinical outcome in pediatric acute myeloid leukemia

While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs. Stem Cells 2015;33:2431–2441

The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed. (C) 2015 The Authors. Published by Elsevier Ltd.

We examined whether ex vivo expansion of umbilical cord blood progenitor cells affected their capacity to generate immune cells such as T lymphocytes (TLs) and dendritic cells (DCs). The capacity to generate TLs from cord blood CD34(+) cells expanded for 14 days (d14) was compared with that of nonexpanded CD34(+) cells (d0) using fetal thymus organ cultures or transfer into nonobese diabetic/severe combined immunodeficient mice. The cell preparations yielded comparable percentages of immature (CD4(+)CD8(-), CD4(+)CD8(+)) TLs and functional mature (CD3(+)CD4(+), CD3(+)CD8(+)) TLs with an analogous TCR (T-cell receptor)-Vbeta repertoire pattern. As regards DCs, d0 and d14 CD34(+) cells also yielded similar percentages of CD1a(+) DCs with the same expression levels of HLA-DR, costimulatory and adhesion molecules, and chemokine receptors. DCs derived from either d14 or d0 CD34(+) stimulated allogeneic TLs to the same extent, and the cytokine pattern production of these allogeneic TLs was similar with no shift toward a predominant Th1 or Th2 response. Even though the intrinsic capacity of d14 CD34(+) cells to generate DCs was 13-fold lower than that of d0 CD34(+) cells, this reduction was offset by the prior amplification of the CD34(+) cells, resulting in the overall production of 15-fold more DCs. These data indicate that ex vivo expansion of CD34(+) cells does not impair T lymphopoiesis nor DC differentiation capacity.

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.

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.

A novel real-time RT-PCR assay for quantification of OTT-MAL fusion transcript reliable for diagnosis of t(1;22) and minimal residual disease (MRD) detection

The most frequent oncogenic activation events characterized in childhood T acute lymphoblastic leukemia (T-ALL) result in the transcriptional activation of genes coding for transcription factors. The main genes are TAL1/SCL, a member of the basic region helix-loop-helix gene family, and HOX11L2, a member of the homeobox-containing protein family. To gain insight into the pathogenesis of this type of hematologic malignancy, we analyzed 28 T-ALL samples. SIL-TAL1/SCL fusion was detected in 6 patients; expression of HOX11L2 was observed in 6 patients and of HOX11 in 3 patients. With one exception, these activations did not occur simultaneously in the same patients, and they allowed the subclassification of 50% of the patients.SIL-TAL1 fusion was detected in association withHOX11 expression in one patient and with a t(8;14) (q24;q11) in another. High expression of LYL1,LMO2, or TAL1 was observed mainly in samples negative for HOX11L2 expression. HOX11L1 andHOX11 expression were observed in one instance each, in the absence of detectable chromosomal abnormality of their respective loci, on chromosomes 2 and 10, respectively. HOX11L2 expression was associated with a chromosome 5q abnormality, the location of theHOX11L2 locus in each case tested. Finally, our data show that HOX11L2 expression was a suitable marker for minimal residual disease follow-up and was significantly associated with relapse (P = .02).

A hundred and eight patients less than 60 years old with de novo acute myeloid leukemia were treated between 1982 and 1994 by protocols including final intensification with a transplant using autologous bone marrow purged by mafosfamide in first remission in the absence of an HLA-matched sibling donor available for allograft. From 1989, we attempted to improve tumor control by using high-dose anthracyclines in induction, by increasing from one to two the number of consolidation courses pre-transplant and by introducing intermediate doses of cytarabine in the first consolidation course. The CR rate was 77% (33/43) before 1989 and 90% (59/65) after 1989 (P = 0.06). Forty-five out of the 59 patients (76%) who achieved CR after 1989 could undergo bone marrow grafting in CR1 vs 16/33 (48%) before 1989 (P = 0.01). In spite of the higher proportion of patients above 50 years after 1989 (32%) toxicity was mild and an adequate graft was obtained more frequently after one collection. The principal factor relating to improvement in graft feasibility was the post-1989 modification of induction and consolidation regimens. This improvement in graft feasibility was associated with a better disease-free survival (DFS) (48 +/- 7% vs 32 +/- 8%, P = 0.04) and overall survival (OS) (53 +/- 6% vs 30 +/- 7%, P = 0.007) at 5 years. By multivariate analysis four factors were associated with overall survival (OS): karyotype, white blood cell count at diagnosis, treatment regimen and bone marrow grafting in CR1. This global approach should be prospectively compared with intensive chemotherapy.

Background: The genetic landscape of adult acute myeloid leukemias (AML) has been recently unravelled. This makes achievable the determination of a comprehensive profile of driver lesions for virtually all patients at diagnosis. Recent studies using multi-target minimal residual disease (MRD) strategies with around 1% sensitivity indicate that the clearance of all molecular events after chemotherapy is associated with better survival. To improve the clono-specificity and the sensitivity of this approach, after a precise determination of AML clonal composition, we combined cytogenetic, FISH, and high sensitivity deep sequencing technologies to monitor the MRD in a series of 69 patients. Methods: Forty-five consecutive patients reflecting the genetic diversity of AML were prospectively included and 24 patients were retrospectively studied. All patients received an anthracycline + cytarabine based regimen. The clonal architecture was established at diagnosis based on NGS-targeted resequencing (122 gene panel) and cytogenetic data. Lesions were next investigated in complete remission (CR). Based on the initial clonal composition, targeted resequencing panels were designed to improve the sensitivity by the use of unique molecular barcodes (Haloplex High Sensitivity, or HS-NGS assay). Cytogenetic events were evaluated by FISH. Results: In the 69 patients, a median of 4 genetic or chromosomal events were identified per patient (range 0-10). One patient had no evaluable target allowing MRD evaluation in 68/69 patients. To determine the threshold of detection of the HS NGS assay, we analyzed the frequency of mutant reads in multiple samples expected to be wild type for 31 given SNVs and 2 indels. A consensus threshold of detection was set at a variant allele frequency (VAF) of 0.2% for all lesions. In CR samples, early initiating events frequently persisted after treatment, especially mutations in DNMT3A, TET2, ASXL1, EZH2, IDH1, TP53, SRSF2, and U2AF1. Mutations in FLT3, NRAS, KIT, NPM1, CEBPA, WT1, IDH2 and BCOR were the most frequently cleared events. Seven patients did not reach CR after one course, and two had no available material after one course. In the 59 remaining patients, we tested whether the global response level of all targets was associated with prognosis. We used the median VAF of the first events of all clonal architectures to separate good responder from poor responders (i.e. VAF = 1.66%). At 2 years, there was a trend to lower leukemia free survival (LFS) probability in poor responders (31.7+/-9.9% vs 51.7+/-9.8%, p=0.08) with no translation in overall survival (OS). We next investigated if the persistence of two or more detectable markers was associated with prognosis. The 58 patients with more than one evaluable event were consequently separated in two groups: patients with 0 or 1 marker above the detection threshold after treatment (n=31), and patients with 2 or more detectable lesions (n=27). At 2 years, DFS was 64.9+/-9.3 % in patients with 0 or 1 detectable marker vs 19.8+/-8.7% in patients with 2 or more detectable markers (p=0.001). OS probability was higher in patients with 0 or 1 detectable marker 84+/-6.6% vs 57.1+/-10.5% (p=0.023). When focusing on the 40 patients with intermediate cytogenetics, persistence of 2 or more markers was associated with lower LFS (57+/-11.8% vs 19.4+/-10.5 p=0.0048) and with a trend to lower OS (85+/-8% vs 61+/-11.9% p=0.07). Similar results were observed when restricting the analyses to the 42 prospectively included patients (At 2 years: LFS 73+/-10% vs 24+/-10%, p=0.0026 and OS 90.2+/-6.6% vs 62.8+/-11.5%, p=0.036). In 50 patients with 3 or more identified events, the persistence of 3 or more markers after one course was associated with a very high risk of relapse (DFS 23.5+/-10.3 % vs 75.8 +/-7.5% at one year, p Conclusion Our study shows the high prognostic value of a personalized multi-target clono-specific MRD evaluation that can be used in nearly all AML patients. Detection of two or more events in more than 0.4% cells after one course is associated with lower survival, in particular in intermediate cytogenetic patients. Larger studies are needed to confirm the results and to evaluate if this strategy could be useful to guide treatment decisions. Disclosures No relevant conflicts of interest to declare.

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

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.

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.

Radiotherapy may induce irreversible damage on healthy tissues surrounding the tumor. It has been reported that the majority of patients receiving pelvic radiation therapy show early or late tissue reactions of graded severity as radiotherapy affects not only the targeted tumor cells but also the surrounding healthy tissues. The late adverse effects of pelvic radiotherapy concern 5% to 10% of them, which could be life threatening. However, a clear medical consensus concerning the clinical management of such healthy tissue sequelae does not exist. Although no pharmacologic interventions have yet been proven to efficiently mitigate radiotherapy severe side effects, few preclinical researches show the potential of combined and sequential pharmacological treatments to prevent the onset of tissue damage. Our group has demonstrated in preclinical animal models that systemic mesenchymal stromal cell (MSC) injection is a promising approach for the medical management of gastrointestinal disorder after irradiation. We have shown that MSCs migrate to damaged tissues and restore gut functions after irradiation. We carefully studied side effects of stem cell injection for further application in patients. We have shown that clinical status of four patients suffering from severe pelvic side effects resulting from an over-dosage was improved following MSC injection in a compationnal situation.

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.

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.

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.

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.

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.