Françoise Norol

Bone Marrow Mesenchymal Stem Cells Suppress Lymphocyte Proliferation In Vitro but Fail to Prevent Graft-versus-Host Disease in Mice

Several reports have suggested that mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect in vitro, and thus may have a therapeutic potential for T cell-dependent pathologies. We aimed to establish whether MSCs could be used to control graft-vs-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation. From C57BL/6 and BALB/c mouse bone marrow cells, we purified and expanded MSCs characterized by the lack of expression of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells. These MSCs suppressed alloantigen-induced T cell proliferation in vitro in a dose-dependent manner, independently of their MHC haplotype. However, when MSCs were added to a bone marrow transplant at a MSCs:T cells ratio that provided a strong inhibition of the allogeneic responses in vitro, they yielded no clinical benefit on the incidence or severity of GVHD. The absence of clinical effect was not due to MSC rejection because they still could be detected in grafted animals, but rather to an absence of suppressive effect on donor T cell division in vivo. Thus, in these murine models, experimental data do not support a significant immunosuppressive effect of MSCs in vivo for the treatment of GVHD.

A common bipotent progenitor generates the erythroid and megakaryocyte lineages in embryonic stem cell-derived primitive hematopoiesis.

The megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA(+)CD41(-)) and MK (GPA(-)CD41(+)) lineages. In contrast to erythropoietin (EPO)-dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.

Influence of clinical status on the efficiency of stored platelet transfusion.

The efficiency of stored platelet transfusion was evaluated in terms of clinical status in 141 thrombocytopenic patients. In a paired prospective study in which fresh platelets were used as controls, clinical efficiency was assessed on the basis of the ability to increase platelet count (recovery) and the time to the next transfusion (D). In 48 clinically stable patients, recovery of fresh and stored platelets was similar (47% and 41%, respectively) and the interval to the next transfusion was D4 and D3. In contrast, 27 patients who had bacterial infections showed significantly different recoveries (24%/5%) and the interval to the next transfusion was D3/D1 for fresh and stored platelets respectively. Similarly, in 16 patients who were treated concurrently with amphotericin B, 18 other patients with graft‐versus‐host disease, nine with splenomegaly and four with veno‐occlusive disease (VOD), fresh platelets performed better than stored platelets, showing recoveries of 27%/18%, 29%/15%, 16%/3% and 15%/2%. Furthermore, the need for retransfusion within 24 h was significantly increased with stored platelets. In 19 patients with anti‐HLA alloimmunization who were transfused with HLA‐matched fresh and stored apheresis platelet concentrate (APC), efficiency was similar (38%/36% and D4/D3). This study indicates that the storage induces an impressive decrease in the in‐vivo platelet recovery and survival in patients with certain clinical conditions.

Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions

The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14(low)CD16(-) precursor to form CD14(high)CD16(+) cells without producing the CD14(high)CD16(-) cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation.

To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short‐term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3‐L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)‐3 (50 ng/ml each); CD34+ cells, granulocyte–macrophage colony‐forming units (GM‐CFU) and megakaryocytic colony‐forming units (MK‐CFU) were amplified, respectively, 10·5‐, 20·5‐ and 17·9‐fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 × 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 × 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 × 109/l) and neutrophil (> 0·5 × 109/l) recovery occurred on days 8 (range 6–9) and 9 (range 6–11), respectively, compared with days 12 (range 10–15) and 14 (range 11–16); platelets recovered (> 20 × 109/l) on day 9 (range 7–12) compared with day 13 (range 11–15) in the control group (P < 0·05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow‐up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long‐term haematopoiesis.

HEPATITIS C VIRUS INFECTION AND ALLOGENEIC BONE MARROW TRANSPLANTATION

Serum antibodies to hepatitis C virus (HCV) were tested for inpatients undergoing allogeneic BMT to determine the risk of acquiring HCV infection and the role of HCV in posttransplant liver complications. The HCV seroconversion rate was evaluated according to the date of BMT and blood donor screening at the time. Anti-HCV antibodies (anti-HCV) were detected with a second-generation ELISA and confirmed with a second-generation radioimmunoblot assay. All patients received leukocyte-depleted blood products and most received apheresis platelet concentrates. One hundred twenty of 181 consecutive patients transplanted from January 1987 to December 1991 were anti-HCV-negative before BMT, had at least 6 months of follow-up, and were thus evaluated for the seroconversion rate. Before screening for non-A, non-B hepatitis, 14% of the patients seroconverted to HCV (0.44% per unit transfused). After introduction of screening for alanine aminotransferase and antibodies to hepatitis B core antigen the risk of seroconversion was 4% per patient (0.26% per unit). When, in addition, blood was screened for anti-HCV the risk fell to 1.6% (0.03% per unit). Positive anti-HCV status before and after BMT was not predictive of veno-occlusive disease, liver graft-versus-host disease (GVHD), or death due to liver dysfunction. In contrast, the risk of chronic hepatitis was significantly increased.

Ex vivo expansion marginally amplifies repopulating cells from baboon peripheral blood mobilized CD34+ cells

Summary.  The ability of ex vivo expansion to increase the long‐term repopulating capacity of a graft is still unknown. One problem is the most reliable way to quantify transplantable cells. We addressed this point in a baboon model based on autologous transplantation of serial limiting doses of non‐manipulated or ex vivo‐expanded mobilized CD34+ cells and determined the threshold doses of non‐manipulated and expanded cells which supported long‐term multilineage engraftment. In the expansion group, CD34+ cells were cultured for 6 d with a combination of early acting cytokines (Flt3‐ligand, stem cell factor, thrombopoietin and interleukin 3). Grafted cells were characterized by their surface antigens and biological properties [semisolid assays, long‐term culture‐initiating cells (LTC‐IC) and non‐obese diabetic severe combined immunodeficient reconstituting cells (SRC)]. Animals were followed for at least 12 months post transplantation. The expansion protocol yielded 12·3‐fold, 16·9‐fold, 3·7‐fold, 3·5‐fold and 2·2‐fold increases in CD34+ cells, granulocyte‐‐macrophage colony‐forming units (CFU‐GM), megakaryocyte CFU (CFU‐MK), LTC‐IC and SRC respectively. It induced a modest increase in the long term reconstitutive ability of the graft; the threshold value for long‐term engraftment was 0·5 × 106/kg CD34+ cells in the control group and 0·3 × 106/kg CD34+ cells in the expansion group, although one animal in this latter group remained hypoplastic. Frequencies of SRC had a high predictive value of long‐term engraftment (r > 0·80). The main advantage of the protocol was the acceleration of granulocyte recovery, achieved at the different doses tested. In conclusion, these experiments suggest that this ex vivo expansion protocol marginally amplifies long‐term reconstituting cells.

Coronary artery spasm after infusion of cryopreserved cord blood cells

Cord blood cells (CBC) from both related and unrelated donors are being used as a source of haematopoietic stem cells (HSC) in a variety of haematological disorders. The advantages of CBC are the immediate availability, the absence of risk to the donor, the potentially reduced risk of GVHD and a lower need for HLA compatibility between the donor and the recipient. Nevertheless, the CBC content and the HLA disparities have a considerable effect on the final outcome of the transplantation. Cryopreservation allows for the long-term storage of CBC. DMSO is the most frequently used cryopreservation agent. However, the use of DMSO has been reported to induce complications ranging from nausea, vomiting, abdominal pain to life-threatening cardiac arrhythmias or cardiopulmonary events, after infusion of cryopreserved BM or peripheral HSC. We report here on a 60-year-old patient having been treated in our department for an atypical chronic myeloid leukemia, bcr/abl negative, with trisomy 8, which was diagnosed in 2003 and was transformed in 2004. The patient received in September 2004, a chemotherapy regimen with daunorubicin, aracytine and cyclosporin, as a modulator of the P-glycoprotein’s activity, and in November 2005, idarubicin and high-dose aracytine. He relapsed on May 2006 and received high-dose aracytine, which permitted a complete cytological and cytogenetic remission. After the consolidation treatment, in July 2006, the BM aspirate smear revealed persistent CR. Pretransplantation echocardiography showed normal cardiac function. The patient received a reduced-intensity conditioning regimen, according to the protocol of Barker et al., consisting of CY (50mg/kg once daily i.v.) on day 6, fludarabine (40mg/m) once daily i.v. for 5 consecutive days (days 6 to 2) and a single dose of 200 cGy of TBI (day 1). The patient underwent a cord blood transplantation from an unrelated female donor (HLA compatibility: 4/6, ABO group Oþ for both donor and recipient). Prevention from GVHD consisted of oral cyclosporin (12.5mg/kg/day) and mycophenolate mofetil (30mg/kg/ day) from day 3. Hepatic venocclusive disease was prevented with i.v. unfractionated heparin 100 IU/kg/day from day 6 until day 14. A single subcutaneous injection of G-CSF (Pegfilgrastim-Neulasta) was administered the day following the transplantation (6mg). Thirty minutes before the transplantation, 50mg of hydrocortisone and 5mg of dexchlorpheniramine were administered intravenously. The administered graft contained 0.30 10/kg CD45þ cells, 0.09 10/kg CD34þ cells and 3.03 10/kg CFU-GM. RBCs have not been depleted before cryopreservation and the product haematocrit was 12.2%. Microbiological controls were negative. The graft was stored in liquid nitrogen in the presence of 10% DMSO, according to the protocol of Rubinstein et al. On the day of transplantation, the bag was thawed in a 371C waterbath. The cryopreservation solution was removed by centrifugation and cells were suspended in a final volume of 175ml with 4% albumin. Graft was given to the patient at a rate of 15ml/min through a central venous catheter. Fifteen minutes after the onset of the transfusion, the patient presented severe abdominal pain, nausea and vomiting, hypertension (180/110mm Hg) and hypothermia (351C). Oxygen saturation was 90%. The electrocardiogram showed ST-segment elevation at V1, V2, V3 and V4 leads and slight ST-segment depression at V5 and V6 leads (Figure 1a). Troponin was slightly elevated (0.9mg/l), whereas myoglobin and creatine kinase were within normal values. The patient was relieved after the inhalation of nitroglycerin and the administration of aspirin (1000mg p.o.), morphine (20mg i.v.) and nasal O2 (2 l/min). After the acute phase, nitroglycerin was administered in continuous i.v. infusion of 1mg/h for 24 consecutive hours. A coronography was urgently effectuated. No arterial occlusion was observed. The echocardiography revealed a slight atrial hypokinesia. The following day, the electrocardiogram showed inverted, deep T-waves at V2, V3, V4, V5 and V6 leads (Figure 1b). Three days later, the electrocardiogram was normalized. The episode was attributed to coronary artery spasm. The patient died several weeks after the transplantation from multiorgan failure. Cardiovascular side effects, such as sinus bradycardia, transient heart blocks or even fatal cardiopulmonary events, have been reported after infusion of cryopreserved HSC. Nevertheless, the pathogenesis of the aforementioned complications is not clear and is supposed to be multi-factorial. Apart from DMSO toxicity, other factors such as cell lysis’ products, toxicity related to previous treatments or conditioning regimens, hypothermia of the infused cells or acute volume expansion, have been implicated in the pathogenesis of cardiovascular complications following the infusion of cryopreserved HSC. Such complications have been usually observed after administration of HSC when thawing without washing procedure. Recently, a French team reported severe neurological accidents after PBSC transplantation related to the number of polymorphonuclear cells in the graft and to high administration rate (F Norol, personal communication). This is the first report of the occurrence of a coronary spasm after the infusion of cryopreserved HSC and, more interestingly, after the infusion of CBC. It seems difficult to Bone Marrow Transplantation (2007) 40, 397–398 & 2007 Nature Publishing Group All rights reserved 0268-3369/07

Regulatory T Cell Content in the Bone Marrow Graft Does Not Predict the Occurrence of Acute GVHD

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Heterozygous and Homozygous JAK2V617F States Modeled by Induced Pluripotent Stem Cells from Myeloproliferative Neoplasm Patients

The subpopulation of regulatory T cells (Treg) was shown to play a key role in alloreactive responses. In allogeneic hematopoietic stem cell transplantation, several groups tested whether Treg content in transplants correlates with graft-versus-host disease (GVHD) with controversial results. In a retrospective study of 49 consecutive HLA-matched sibling transplantations, we studied the relationship between Treg content in bone marrow transplants and acute GVHD (aGVHD) occurrence. We observed a large variability in Treg in bone marrow grafts. However, contrary to previous observations in peripheral blood stem cells transplantation, we report that the Treg content of allogeneic bone marrow transplantation did not predict the occurrence of aGVHD.