Jean-Francois Lambert

Akt Signaling Regulates Side Population Cell Phenotype via Bcrp1 Translocation

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp-/- mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.

Quick sex determination of mouse fetuses.

We designed a rapid, simple and accurate PCR method to determine sexual identity of mouse fetuses collected on embryonic day 15. A multiplex PCR amplification was used to detect male-specific sequence (Sry) in DNA extracted from fetal livers through SDS denaturation followed by high salt extraction and precipitation. This extraction method resulted in sufficiently purified DNA in < 1 h and was suitable for PCR. The DNA obtained was amplified using a robot thermal cycler for 33 cycles. The reaction was performed in 50 microl, using two sets of primers specific for Sry gene (chromosome Y) and IL3 gene (chromosome 11). Amplification duration was 1.5 h. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The 402 bp band (Sry) obtained identifies the male fetuses and the 544 bp product (IL3) confirms the correct amplification of the template DNA. The entire procedure took < 4 h. The specificity of the method was confirmed by fluorescent in situ hybridization using a specific male probe on cultured male and female neural stem cells. This method allowed the preparation and culture of pure male and female neural stem cells from fetal tissue.

Marrow Stem Cells Shift Gene Expression and Engraftment Phenotype with Cell Cycle Transit

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3′ end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from “stem cell genes” being highly expressed at 0 h and turned off at 48 h, while “cell division” genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

Homing of Purified Murine Lymphohematopoietic Stem Cells: A Cytokine-Induced Defect

This study was designed to establish a direct homing assay using purified lineage-negative Sca-1-positive (Lin(-) Sca(+)) murine bone marrow cells and to evaluate the effects of cytokines on homing. C57BL/6 Lin(-) Sca(+) marrow stem cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and then injected by tail vein into untreated C57BL/6 mice. Marrow was harvested at various times after cell infusion and analyzed on a high-speed MoFlo cell sorter for fluorescent positive events, using a large event analysis, with at least 16 million total events analyzed. We have shown that homing of Lin(-) Sca(+) cells plateaus by 1 h, and at 3 h post-infusion is linear between 50,000 and 1,000,000 infused cells. This forms a base for a homing assay in which 250,000 CFDA-SE labeled Lin(-) Sca(+) marrow cells are infused and then recovered from marrow 3 h later, followed by a large-event fluorescence-activated cell sorting (FACS) analysis. We found that 7.45-9.32% of infused cells homed and that homing of stem cells cultured for 48 h in interleukin-3 (IL-3), IL-6, IL-11, and steel factor cultured cells was defective when compared to noncultured cells. Exposure of marrow stem cells to IL-3, IL-6, IL-11, and steel factor induces a stem cell homing defect, which probably underlies the engraftment defect previously characterized under these conditions.

Intrinsic hematopoietic stem cell/progenitor plasticity: Inversions

Traditional concepts indicate that stem cells give rise to progenitor cells in a hierarchical system. We studied murine engraftable stem cells (ESCs) and progenitors in in vitro and found that ESC and progenitors exist in a reversible continuum, rather then a hierarchy. B6.SJL and BALB/c marrow cells were serially cultured with thrombopoietin (TPO), FLT‐3 ligand (FLT‐3L), and steel factor through cell cycle. Progenitors (high‐proliferative potential colony‐forming cells (HPP‐CFC) and colony‐forming unit culture (CFU‐c)) and ESC capacity was determined. The cell cycle status of purified lineagenegativerhodaminelowHoechstlow stem cells was determined under the same conditions using tritiated thymidine incorporation and cell counts. We found an inverse relationship between progenitors and ESC, which occurred during the first cell cycle transit and was reversible. We have termed these progenitor/stem cell inversions and found that these inversions were consistently seen at 28–32 h of culture, representing early S‐phase. We observed 13 major reversible increases in progenitor numbers from one time‐point to another during the first cell cycle transit; this was coupled with 11 major ESC decreases and in 2 instances ESC were at baseline. These studies indicate that primitive marrow cells reversibly shift from ESC to progenitors without differentiation occurring. They exist as a fluctuating continuum. J. Cell. Physiol. 199: 20–31, 2004© 2003 Wiley‐Liss, Inc.

Murine allogeneic in vivo stem cell homing

Stem cell homing has been studied in syngeneic models and appears to be rapid (<1 h) and dependent on cellular adhesion and migration factors. We utilized a full H2‐mismatched transplantation model to determine the basics of allogeneic homing. C57BL/6J Lin‐Sca‐1+ cells were labeled with CFSE and injected in non‐myeloablated BALB/c mice. Fluorescent cell detection was via high‐speed FACS analysis. Alternatively, B6.SJL whole bone marrow cells were injected in lethally irradiated BALB/c mice (10 Gy). One, 3, 6, and 24 h after transplant, marrow was harvested and cells were either plated for high proliferative potential colony‐forming cell (HPP‐CFC) assay or secondarily injected into myeloablated (8 Gy) C57BL/6J mice using 10% competing C57BL/6J marrow. Chimerism was evaluated at 8 weeks. CFSE+ cells were detected in the bone marrow 1, 3, and 6 h after injection. The numbers were moderately lower when compared to syngeneic homing possibly due to strain effect. Conversely, utilizing a surrogate or secondary assay, we observed a decline of secondary engraftment of harvested cells over time, but not of HPP‐CFC. Combining experiments and normalizing the 1‐h time point to 100% (to allow comparison), we observed a mean relative engraftment of 87 ± 29%, 72 ± 21%, 84 ± 35% of the 1 h level at 3, 6, and 24 h respectively. HPP‐CFC assay showed no significant variation as a homing surrogate over 1–6 h. These data indicate a rapid homing into allogeneic recipients with a plateau at 1 h. The decline of secondary engraftability over time may indicate a phenotype alteration of homed cells. J. Cell. Physiol. 211: 386–391, 2007.

Long-term follow-up of European APL 2000 trial, evaluating the role of cytarabine combined with ATRA and Daunorubicin in the treatment of nonelderly APL patients.

All‐trans retinoic acid (ATRA) combined to anthracycline‐based chemotherapy is the reference treatment of acute promyelocytic leukemia (APL). Whereas, in high‐risk patients, cytarabine (AraC) is often considered useful in combination with anthracycline to prevent relapse, its usefulness in standard‐risk APL is uncertain. In APL 2000 trial, patients with standard‐risk APL [i.e., with baseline white blood cell (WBC) count <10,000/mm3] were randomized between treatment with ATRA with Daunorubicin (DNR) and AraC (AraC group) and ATRA with DNR but without AraC (no AraC group). All patients subsequently received combined maintenance treatment. The trial had been prematurely terminated due to significantly more relapses in the no AraC group (J Clin Oncol, (24) 2006, 5703–10), but follow‐up was still relatively short. With long‐term follow‐up (median 103 months), the 7‐year cumulative incidence of relapses was 28.6% in the no AraC group, compared to 12.9% in the AraC group (P = 0.0065). In standard‐risk APL, at least when the anthracycline used is DNR, avoiding AraC may lead to an increased risk of relapse suggesting that the need for AraC is regimen‐dependent. Am. J. Hematol. 88:556–559, 2013.

Engraftment of post 5-fluorouracil murine marrow into minimally myeloablated (100 cGy) murine hosts.

Minimal myeloablative approaches are now being widely applied in the treatment of different hematological malignancies. One hundred cGy whole-body irradiation is a stem-cell-toxic, relatively non-myelotoxic treatment that allows for relatively high levels of donor chimerism. 5-Fluorouracil (5-FU) treatment leads to a relative concentration of high proliferative potential-colony-forming cell (HPP-CFC) and is an approach that has been used to induce in vivo progenitor/stem cell cycling to facilitate retroviral integration in gene therapy approaches. We have now evaluated the capacity of marrow harvested 1, 2, 6, or 12 days after 5-FU treatment (150 mg/kg) to engraft in 100 cGy-treated female BALB/c mice. Engraftment was assessed at 3, 10, and 24 weeks. A rapid induction of an engraftment defect occurred 1 day post 5-FU and persisted through day 6 with a recovery by day 12. To evaluate cell cycle status of normal and 5-FU-treated marrow cells, male donors received hydroxyurea (900 mg/kg i.v.) or phosphate-buffered saline (PBS), 2 h prior to marrow harvest and transplantation into submyeloablated female recipients. Engraftment levels were similar for hydroxyurea-treated mice and controls. Thus, these studies show transiently defective engraftment of 5-FU-treated marrow into submyeloablated hosts, which may be related to the cell cycle status of the stem cells.

Hematopoietic stem cells in microgravity

Abstract Engraftment and progenitor expansion was compared after microgravity or conventional culture. Rotating Wall Vessels (RWV) were compared with Teflon flask cultures. Two systems were used: 1) Male BALB/c whole bone marrow was cultured with varying cytokines, time intervals ranging from 24 hours to 7 days at 10–40 RPM. The cultured marrow was competed with an equal amount of fresh female marrow into lethally ablated female BALB/c mice. 2) Male B6.SJL (CD45.1) marrow was cultured with varying time intervals ranging from 24 hours to 14 days at 10 RPM with FLT-3 ligand, steel factor, and thrombopoietin. Fresh C57BL/6J (CD 45.2) marrow (3 × 10 6 cells) was competed with 3 × 10 6 cultured B6SJL male cells in lethally ablated male C57BL recipients. Engraftment was analyzed at 3–10 weeks. Progenitor expansion increased over controls by 24 hours with significantly greater expansion in the RWV cultures by 48 hours. The dominant theme was a fluctuation in engraftment phenotype, which transcended cytokine combination and murine strain. A nadir of engraftable stem cells occurred between 36–44 hours in Teflon flask cultures and 30–48 hours in RWV cultures. There was a consistent phase shift of engraftment between culturing conditions of 4–6 hours. The engraftable stem cells in RWV showed superior support of short-term engraftment in multiple experiments up to 32 hours in culture. Slower rational speed improved RWV engraftment and in-vitro progenitor number. Longer culturing time gave similar engraftment results. Surrogate progenitors from both culture conditions did not reflect engraftment data, with an overall increase in progenitors over time compared with dramatic engraftment fluctuation. Enhanced plating efficiency and increased number of progenitors seen in the RWV cultures when compared with the Teflon flask cultures persisting after 24 hours in culture. The fluctuating engraftment phenotype persists with different strains and cytokine combinations, signifying that this is a crucial physiologic phenomenon.

In vivo allogeneic homing using cfse labeled murine stem cells or surrogate secondary transplant

Abstract In order to characterize homing of allogeneic murine stem cells, a direct and a surrogate approach were utilized. First, 105 CFSE-labeled B6/SJL Lin-Sca1+ cells were injected in non-myeloablated BALB/c mice and fluorescent mononuclear cells were detected 1 and 6 hours after transplant by high-speed FACS analysis. In the surrogate approach, 108 B6/SJL whole BM cells were injected in lethally irradiated BALBc mice (10Gy) (5/time point). 1, 3 and 6 hours after transplant, BM was harvested and 1.1×107 cells were secondarily injected into myeloablated (8Gy) C5/BL6 mice (12/time point) with 1.1×107 competing C57/BL6 BM cells. Direct approach—CFSE positive cells were detected by FACS in BM 1 and 6 hours after injection. ∼11×106 events were analyzed in 4 mice. Surrogate approach—Secondary engrafted B6/JL Ly5.1 cells were detected in BM, spleen and thymus of myeloablated C57/BL6 recipient. Percentages of Ly5.1 cells over total Ly5.1 + Ly5.2 cells are shown in the figure. In conclusion, allogeneic homing was detectable from 1 to 6 hours after injection using either a direct approach with purified Lin-Sca1+ cells or a surrogate approach with whole bone marrow.