1. A defect in hematopoietic stem cell migration explains the nonrandom X-chromosome inactivation in carriers of Wiskott-Aldrich syndrome
by Catherine Lacout, Elie Haddad, Siham Sabri, Fedor Svinarchouk, Loic Garçon, Claude Capron, Adlen Foudi, Rym Mzali, Scott B. Snapper, Fawzia Louache, William Vainchenker, and Dominique Duménil
Blood
Volume 102(4):
August 15, 2003
©2003 by American Society of Hematology

2. Reduced homing capacity of WASP-deficient bone marrow cells as compared with wild-type cells.
Reduced homing capacity of WASP-deficient bone marrow cells as compared with wild-type cells. Lethally irradiated female mice received transplants of a mixture of 10 × 106 wild-type and 10 × 106 WASP-deficient bone marrow cells. The bone marrow cells were stained with 2 different dyes: wild-type mice were stained with CSFE, and WASP-deficient cells with PKH26, or vice versa. (A) At 24 hours after the graft, bone marrow and spleen were harvested from each recipient, and nucleated cells were analyzed by flow cytometry to evaluate the proportion of red and green cells in the 2 hematopoietic tissues. Results shown are means ± SD of 4 experiments done with 3 mice in each group. (B) Bone marrow and spleen cells of recipient mice were used for progenitor assays, and 20 colonies per mouse were picked and monitored by Y and neo PCR to determine the origin of the cell that gives rise to the colony. Seven recipient mice were analyzed, and 40 colonies per mouse were harvested (20 from the bone marrow and 20 from the spleen; a total of 140 colonies from the bone marrow and 140 from the spleen were analyzed). Results shown are means ± SD of 7 analyzed mice.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Comparison of progenitor numbers in wild-type and WASP-deficient bone marrow.
Comparison of progenitor numbers in wild-type and WASP-deficient bone marrow. The number of progenitors is the same in the bone marrow of wild-type and WASP-deficient mice. Bone marrow cells from wild-type (gray column) or WASP-deficient (black column) mice were cultured in methylcellulose in the presence of IL-3, TPO, and EPO. After 8 days at 37°C in an atmosphere of 5% CO2 in air, the erythroid, megakaryocytic, and granulo-macrophagic colonies were counted under an inverted microscope. Results shown are mean ± SD of 3 independent experiments.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Time course of Cdc42 activation following SDF-1 stimulation.
Time course of Cdc42 activation following SDF-1 stimulation. Cdc42 activation was studied after SDF-1 (100 ng/mL) activation of Lin– cells from 129Sv and WASP-deficient mice by a pull-down assay. The GST-WASP-CRIB–binding domain was used to evaluate GTP-bound Cdc42 (upper panel). The immunoblot of total lysates by Cdc42 antibody is shown in the lower panel. The last line represents the pull-down and total Cdc42 lysates of transfected cells by a dominant activated Cdc42 (CDc42-Val12).
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

5. Expression of VLA-4 and VLA-5 in Lin–Sca+c-kit+ cells.
Expression of VLA-4 and VLA-5 in Lin–Sca+c-kit+ cells. Lin– cells were first purified by depletion of Lin+ cells. Lin– cells were labeled with antibodies against Sca-1, c-kit, VLA-4, VLA-5, or their isotype controls. VLA-4 expression was studied with an FITC-conjugated antibody (panel A); VLA-5 was studied with a PE-conjugated antibody (panel B). Black indicates 129Sv; gray, WASP-deficient; plain line, specific antibody; dotted line, isotype control.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Effect of collagen I on polarization and actin reorganization.
Effect of collagen I on polarization and actin reorganization. Collagen I induced polarization and actin reorganization in SDF-1–pretreated Lin– 129Sv but not in WASP-deficient cells. Lin– cells from 129Sv and WASP-deficient mice (panels A-B) were either pretreated (panels B,D) or not (panels A,C) by SDF-1 for 10 minutes, allowed to adhere to collagen I–coated coverslips for 1 hour at 37°C, and processed for actin staining by rhodamine phalloidin as described in “Materials and methods.” Scale bar equals 10 μm.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

7. Animal survival and chimerism of animals after bone marrow transplantation (BMT).
Animal survival and chimerism of animals after bone marrow transplantation (BMT). Male mice were used as bone marrow donors. Female recipient mice were letally irradiated, and 3 million nucleated cells were injected intravenously. The mice were killed 2 months after the graft, and the cells from one leg were injected into a new irradiated female mouse. This process was repeated 7 times. (A) Cumulative survival after serial BMT. Each group included 10 mice. The donor marrow from one leg of the previous transplant was individually injected into a new recipient. There was no mortality during the first 3 transplantations. Results of the fourth to seventh transplantations are shown here. (B) Numbers of nucleated cells per leg. At each transplantation, one leg was harvested from each mouse and cells were enumerated; results shown are the means ± SD for the 2 groups after the fourth to the sixth transplantation. (C) Numbers of colony-forming cells (CFCs) per leg at the fourth to the sixth transplantation. (D) Percentage of donor cells observed in recipient mice after each transplantation. At each transplantation, bone marrow cells from recipient mice were cultured in methylcellulose. Twenty colonies per mouse were picked, and monitored by Y and neo PCR.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

8. No defect in self-renewal in stem cells from WASP-deficient mice.
No defect in self-renewal in stem cells from WASP-deficient mice. Long-term bone marrow cultures were established on MS-5 feeder layer with bone marrow cells from wild-type and WASP-deficient mice. First, 3 × 106 bone marrow cells were seeded into 10 mL culture medium in a 25-cm2 flask. Half of each culture was removed every week and replaced with fresh medium. A flask of each culture (one from wild-type and one from WASP-deficient cells) was stopped every week. Nucleated cells were counted and a progenitor assay was performed. No differences were found between the numbers of nucleated cells or clonogenic progenitors of wild-type and WASP-deficient cultures.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology

9. Percentage of progenitor cells from bone marrow or fetal liver of heterozygous female expressing normal (X+) or mutated (X–) allele.
Percentage of progenitor cells from bone marrow or fetal liver of heterozygous female expressing normal (X+) or mutated (X–) allele. Bone marrow and fetal liver cells from heterozygous females were seeded into methylcellulose in the presence or absence of G418. Only the cells expressing the X-chromosome carrying the mutated WASP gene are able to grow in the presence of G418. Fetal liver cells from heterozygous females were also injected intravenously into irradiated recipient mice, and at 24 hours or 3 months after the graft, bone marrow cells were harvested and cultured in methylcellulose in the presence or absence of G418. At 7 days later, the number of colonies were scored. Results are expressed as the percentage of clonogenic progenitors expressing the normal (X+) or the mutated (X–) allele. This proportion was compared (by Wilcoxon test and χ2 test) to the proportion 50:50 that is observed when random X-chromosome inactivation occurs. As this skewed X inactivation is observed 24 hours after transplantation, this indicates that hematopoietic progenitors expressing WASP have a selective advantage over the WASP-deficient progenitors in homing ability. Error bars indicate SD.
Catherine Lacout et al. Blood 2003;102:
©2003 by American Society of Hematology