Plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion.

Sickle cell disease is characterized by chronic anemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Hematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected hematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and properties of the red blood cells hamper the harvesting and immunoselection of patients' stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of hematopoietic stem and progenitor cells into the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients undergoing red blood cell exchange to decrease the hemoglobin S level to below 30%. The secondary objective was to measure the efficiency of mobilization and isolation of hematopoietic stem and progenitor cells. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of hematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize hematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Clinicaltrials.gov identifier: NCT02212535.

Faster T-cell development following gene therapy compared with haploidentical HSCT in the treatment of SCID-X1.

During the last decade, gene therapy via ex vivo gene transfer into autologous hematopoietic stem cells has emerged as a convincing therapy for severe combined immunodeficiency caused by ILR2G mutation (SCID-X1) despite the occurrence of genotoxicity caused by the integration of first-generation retroviral vectors. However, the place of gene therapy among the therapeutic armamentarium remains to be defined. We retrospectively analyze and compare clinical outcomes and immune reconstitution in 13 consecutive SCID-X1 patients having undergone haploidentical hematopoietic stem cell transplantation (HSCT) and 14 SCID-X1 patients treated with gene therapy over the same period at a single center level: the Necker Children's Hospital (Paris, France). Our results show a clear advantage in terms of T-cell development of gene therapy over HSCT with a mismatched donor. Patients treated with gene therapy display a faster T-cell reconstitution and a better long-term thymic output. Interestingly, this advantage of gene therapy vs haploidentical HSCT seems to be independent of the existence of clinical graft-versus-host disease in the latter condition. If data of safety are confirmed over the long term, gene therapy for SCID-X1 appears to be an equal, if not superior, alternative to haploidentical HSCT.

Genotoxic signature in cord blood cells of newborns exposed in utero to a Zidovudine-based antiretroviral combination.

BACKGROUND: The genotoxicity of zidovudine has been established in experimental models. The objective of the study was to identify genotoxicity markers in cord blood cells from newborns exposed in utero to antiretroviral (ARV) combinations containing zidovudine. METHODS: Cells were investigated by karyotyping and gene expression analysis of the CD34(+) hematopoietic stem/progenitor cell (HPC) compartment. RESULTS: Karyotyping of the cord blood cells from 15 ARV-exposed newborns and 12 controls revealed a higher proportion of aneuploid cells in the exposed group (median, 18.8% [interquartile range, 10.0%-26.7%] vs 6.6% [interquartile range, 3.1%-11.7%]; P < .001). All chromosomes were involved, with a random distribution of these alterations. Gene expression profiling of CD34(+) HPCs from 7 ARV-exposed and 6 control newborns revealed that >300 genes were significantly upregulated or downregulated by at least 1.5-fold in the exposed group (P < .05 for all comparisons). Significant alterations of genes involved in cell cycle control, mitotic checkpoints, and DNA repair were identified. Although this study does not allow discrimination between the roles of each of the 3 drugs, both cytogenetic and transcriptional findings are similar to those in cellular experiments that used zidovudine alone. CONCLUSIONS: The cord blood cells, including hematopoietic stem cells, from newborns exposed in utero to a zidovudine-based ARV combination present cytogenetic and transcriptional abnormalities compatible with DNA damage.

Human T-lymphoid progenitors generated in a feeder-cell-free Delta-like-4 culture system promote T-cell reconstitution in NOD/SCID/γc(-/-) mice.

Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34(+) CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc(-/-) (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αß T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting.

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro.

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56(+) cells grew rapidly, a population of CD15(+) cells emerged, partly from CD56(+) cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56(+) and CD15(+) cells shared osteogenic and chondrogenic abilities, while CD56(+) cells presented a myogenic capacity and CD15(+) cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

Bone marrow microenvironment in fanconi anemia: a prospective functional study in a cohort of fanconi anemia patients.

Fanconi anemia (FA) is a rare condition due to the genetic inactivation of the FA/BRCA pathway. During childhood, most FA patients display progressive bone marrow failure (BMF), the mechanism of which has not been clarified to date. We analyzed BM mesenchymal stem cells (MSCs) from a series of 20 FA patients with BMF (patient median age 12.5 years old, range 7-34). Expression of FANCD2 and sensitivity to mitomycin C, differentiation capacities, and hematopoiesis-supporting abilities, as well as proliferation, cell senescence, and telomere length were assessed. FA MSCs demonstrated hypersensitivity to mitomycin C compared to control MSCs, as expected for FA cells. FA MSCs had normal immunophenotype, support long-term culture of hematopoietic stem cells (HSCs), and display normal differentiation capacities. Telomere loss during cell aging was similar for FA and control MSCs. However, FA MSCs showed reduced long-term proliferation ability, higher stem cell factor and interleukin-6 levels, and increased expression of senescent-associated beta-galactosidase compared to normal MSCs, suggesting a potential role of the BM microenvironment in long-term BMF.

Quantification of nucleated red blood cells in allogeneic marrow graft and impact of processing on recovery.

BACKGROUND: In allogeneic bone marrow transplantation (BMT), a higher nucleated and CD34+ cell dose has been reported to improve various outcomes. Other cell types, such as lymphocyte subsets, also influenced BMT results. While nucleated red blood cells (NRBCs) represent a subset of bone marrow (BM) cell subpopulation, the question of their quantification in BM grafts and the impact of BM processing on their recovery has not been addressed. STUDY DESIGN AND METHODS: In a prospective study on 77 BM products, NRBCs were enumerated by flow cytometry and the recovery analyzed after manipulation. Because NRBCs could compromise white blood cell count, the impact of NRBC count on CD34+ cell percentage and total nucleated cell (TNC) dose were also determined. RESULTS: The mean percentage of NRBCs in BM grafts was 21.6 percent (range, 7.8%-40.9%). Mean NRBC recoveries after BM concentration or RBC depletion were 98.4 and 28.7 percent, respectively, close to those obtained for TNC cells (88.6 and 31.3%, respectively). When corrected with NRBC count, the mean percentages of corrected CD34+ cell and TNC dose were significantly modified when compared with uncorrected values, whatever the type of BM manipulation. CONCLUSION: Our data show that NRBC quantification might be of importance to improve quality control of BM products and to evaluate the influence of NRBCs cell dose on outcomes after BMT.

Ex vivo generation of mature and functional human smooth muscle cells differentiated from skeletal myoblasts.

We described the ex vivo production of mature and functional human smooth muscle cells (SMCs) derived from skeletal myoblasts. Initially, myoblasts expressed all myogenic cell-related markers such as Myf5, MyoD and Myogenin and differentiate into myotubes. After culture in a medium containing vascular endothelial growth factor (VEGF), these cells were shown to have adopted a differentiated SMC identity as demonstrated by alphaSMA, SM22alpha, calponin and smooth muscle-myosin heavy chain expression. Moreover, the cells cultured in the presence of VEGF did not express MyoD anymore and were unable to fuse in multinucleated myotubes. We demonstrated that myoblasts-derived SMCs (MDSMCs) interacted with endothelial cells to form, in vitro, a capillary-like network in three-dimensional collagen culture and, in vivo, a functional vascular structure in a Matrigel implant in nonobese diabetic-severe combined immunodeficient mice. Based on the easily available tissue source and their differentiation into functional SMCs, these data argue that skeletal myoblasts might represent an important tool for SMCs-based cell therapy.

Association of bone marrow natural killer cell dose with neutrophil recovery and chronic graft-versus-host disease after HLA identical sibling bone marrow transplants.

Allogeneic bone marrow (BM) transplant (BMT) outcomes have been correlated with the infused nucleated, CD34(+), and T- cell dose. The potential impact of natural killer (NK) BM infused cell dose has however not been established. We analysed the outcomes of 78 patients receiving an HLA identical BMT. A higher NK cell dose was associated with the speed of neutrophil (P = 0.05) and platelet recovery (P = 0.04). Higher nucleated cells, CD34(+), CD3(+), CD3(+)/4(+), CD3(+)/8(+) and NK cell dose were associated with a lower incidence of chronic GvHD (cGvHD) in univariate analysis. In multivariate analysis, the risk of cGvHD was increased by a lower NK cell dose [hazard ratio (HR) = 2.3 (1.2-4.4) for cell dose <0.9 x 10(6)/kg; P = 0.01] and an older age [HR = 1.4 /10 years (1.1-1.8); P = 0.002]. In addition, a higher CD3(+)/4(+) and NK cell dose were associated with a decreased incidence of viral infections (P = 0.03 and P = 0.06 respectively). No specific cell subpopulation infused dose was associated with survival. In conclusion, a higher BM NK cell dose is associated with an increased speed of neutrophil recovery and a decreased incidence of cGvHD.

ABO-mismatched marrow processing for transplantation: results of 114 procedures and analysis of immediate adverse events and hematopoietic recovery.

BACKGROUND: Red cell (RBC) depletion is needed to bypass ABO mismatch in allogeneic bone marrow transplantation (BMT). Technical and clinical data obtained after bone marrow (BM) processing with a continuous-flow cell separator (Cobe Spectra, Gambro BCT) are reported. STUDY DESIGN AND METHODS: RBC depletion and recovery of nucleated cells, CD3+ cells, CD34+ cells, and colony-forming unit-granulocyte-macrophage were calculated. Bacteriologic contaminations, side effects of graft infusion, and hematopoietic recovery were analyzed. RESULTS: A total of 114 BM samples were processed. The mean volume collected was 1099 mL (range, 390-2450 mL). Initial and residual mean RBCs volumes were 309.9 and 4.0 mL corresponding to a depletion of 98.6 +/- 0.78 percent. Before processing, the mean numbers of nucleated cells, granulocytes, CD3+ cells, CD34+ cells, and CFU-GM were 20.28 x 10(9), 12.79 x 10(9), 1.96 x 10(9), 356.7 x 10(6), and 195.6 x 10(5), respectively. The mean corresponding recoveries after processing were 33.66, 48.98, 82.02, 82.2, and 93.9 percent. Limited side effects were observed in 14 patients without correlation with residual RBCs volume. All but two patients engrafted. CONCLUSION: BM processing with the Cobe Spectra cell separator provides high rates of RBC depletion without significant side effects after BMT.