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HEMATOPOIESIS AND STEM CELLS
The galactocerebrosidase enzyme contributes to the maintenance of a functional
hematopoietic stem cell niche
Ilaria Visigalli,1,2 Silvia Ungari,1,2 Sabata Martino,3 Hyejung Park,4 Martina Cesani,1 Bernhard Gentner,1 Lucia Sergi Sergi,1
Aldo Orlacchio,3 Luigi Naldini,1,2 and Alessandra Biffi1
1San
Raffaele Telethon Institute for Gene Therapy and 2Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milan, Italy; 3Department of
Experimental Medicine and Biochemical Science, Perugia University, Perugia, Italy; and 4Schools of Biology, Chemistry and Biochemistry and the Petit Institute
for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta
The balance between survival and death in
many cell types is regulated by small
changes in the intracellular content of bioactive sphingolipids. Enzymes that either produce or degrade these sphingolipids control this equilibrium. The findings here
described indicate that the lysosomal galactocerebrosidase (GALC) enzyme, defective
in globoid cell leukodystrophy, is involved
in the maintenance of a functional hematopoietic stem/progenitor cell (HSPC) niche
by contributing to the control of the intracellular content of key sphingolipids. Indeed,
we show that both insufficient and supraphysiologic GALC activity—by inherited genetic deficiency or forced gene expression
in patients’ cells and in the disease model—
induce alterations of the intracellular con-
tent of the bioactive GALC downstream
products ceramide and sphingosine, and
thus affect HSPC survival and function and
the functionality of the stem cell niche.
Therefore, GALC and, possibly, other enzymes for the maintenance of niche functionality and health tightly control the concentration of these sphingolipids within HSPCs.
(Blood. 2010;116(11):1857-1866)
Introduction
The lysosomal galactocerebrosidase (GALC) enzyme, whose inherited deficiency results in the lysosomal storage disorder (LSD)
globoid cell leukodystrophy (GLD), catalyzes the hydrolysis of
galactose from several glycosphingolipids, including galactosylceramide (Gal-Cer) and galactosylsphingosine (psychosine; Psy),
which are important for myelination in the nervous system. In
GLD, pathology is characterized by accumulation of nonmetabolized substrates leading to widespread demyelination and patients’
early death1 in the absence of overt abnormalities affecting the
hematopoietic and immune systems. Alterations in the immune
system have been recently described in the Twitcher mouse, a
naturally occurring Galc mutant carrying a stop codon that
abrogates enzyme activity. This defect was attributed to a progressive autonomic denervation of the lymphoid organs of affected
mice.2 The sympathetic nervous system also regulates hematopoietic stem cell (HSC) egress from the bone marrow (BM) niche
upon granulocyte colony-stimulating factor (GCSF) stimulation, as
shown in mice defective in galactosyltransferase (CGT),3 an
enzyme that synthesizes Gal-Cer by the addition of UDP-galactose
to ceramide (Cer). Interestingly, these 2 lipids, as well as other
molecules found in the GALC metabolic pathway, such as sphingosine (Sph) and sphingosine-1-phosphate (S1P), have been reported to regulate cell growth, differentiation, senescence, and
apoptosis in several cell types and are comprised among “bioactive
sphingolipids,”4 small quantitative changes of which can result in
functional consequences in most cell types.
Compared with the other lysosomal enzymes, which are
constitutively expressed in most tissues, GALC protein is found at
low levels in all tested tissue samples.5 When the human GALC
cDNA was cloned, very low GALC expression was obtained
following transfection.6 Moreover, a suboptimal initiation start site
and inhibitory sequences in the 5⬘ regulatory region characterize
the human GALC gene.7,8 Overall, these features suggest the need
for tight physiologic regulation of GALC expression and activity.
We obtained indication of a requirement for such a regulation of
GALC expression within the hematopoietic stem/progenitor cell
(HSPC) compartment in the context of gene therapy studies (B.G.,
I.V., H. Hiramatsu, E. Lechman, S.U., A. Giustacchini, G. Schira,
M. Amendola, S.M., A.O., J. E. Dick, L.N., and A.B., Identification
of HSC-specific miRNAs enables tight regulation of gene expression and effective gene therapy of globoid leukodystrophy, manuscript submitted).
We here investigate the biochemical and functional consequences of alterations in the physiologic GALC expression levels
in HSPCs and show that both insufficient and supraphysiologic
GALC activity is associated to an altered content of bioactive
sphingolipids and to functional abnormalities affecting HSPCs and
their niche. Thus, these data suggest a novel role for GALC in the
maintenance of a healthy hematopoietic niche. Regulation of the
intracellular content of bioactive sphingolipids such as Cer may
represent the mechanism by which the enzyme exerts this activity.
Submitted November 25, 2009; accepted May 17, 2010. Prepublished online as
Blood First Edition paper, May 28, 2010; DOI 10.1182/blood-2009-12-256461.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
Methods
Flow cytometric analysis
For immunostaining, cells were blocked in phosphate-buffered saline
(PBS), 5% rat serum, 2% fetal bovine serum (FBS) for 15 minutes at 4°C,
stained with specific antibody for 20 minutes at 4°C, and washed. Cells
were resuspended in PBS 2% FBS and read on a Becton Dickinson next
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BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
VISIGALLI et al
generation flow cytometer equipped with 3 lasers (Canto II; BD Biosciences). Fluorochrome compensation was performed manually based on
single-color marked samples and/or compensation beads (BD Biosciences)
when appropriate. DIVA software was used for acquisition (BD Biosciences), and data files were exported in the FCS 3.0 format. FlowJo
version 8.5 software (TreeStar) was used for the analysis. All gates were set
based on specific fluorescence minus one (FMO) controls. The gating
strategy is described in supplemental Table 1 (available on the Blood Web
site; see the Supplemental Materials link at the top of the online article).
GLD BM (as approved by the Ethical Committee of the Fondazione San
Raffaele and of the San Gerardo Hospital, Monza, and collection upon
informed consent in accordance with the Declaration of Helsinki) and
transduced with LV at MOI 100 as described.13 BM HSPCs were
transduced using a different cytokine cocktail (IL-3 60 ng/␮L, thrombopoietin [TPO] 100 ng/␮L, SCF 300 ng/␮L, Flt3-L 300 ng/␮L) in CellGro
medium (Cell Genics).
Lentiviral vector production and titration
Lineage⫺ HSPCs were isolated from 4-week-old mice and transduced with
PGK.GFP LV, as described above. After transduction, cells were washed
and transplanted into 8-day-old lethally irradiated mice. Mice were
euthanized 20 hours after transplantation, and BM was harvested for
cytofluorimetric analysis.
Vesicular stomatitis virus (VSV), pseudotyped lentiviral vectors (LV) were
produced by transient cotransfection of the transfer constructs (pRRLsin.
cPPT.humanPosphoGlycerateKinase.murineGalc.Wpre [PGK.Galc]; pRRLsin.
cPPT.humanPGK.GreenFluorescentProtein.Wpre [PGK.GFP]; pRRLsin.
cPPT.humanPGK.humanArylsulfataseA.Wpre [PGK.ARSA]; pRRLsin.cPPT.
murineGalc.minimalCitomegaloVirus-PGK.9humanAcidCeramidase.Wpre
[Galc&AC]; pRRLsin.cPPT.humanGALC.murineGalc.Wpre [GALC.Galc];
pRRLsin.cPPT.humanPGK.DeletedNerveGrowthFactorReceptor.Wpre [PGK.
DeltaNGFR]) and the packaging constructs in 293T cells and titrated by end
point integration in HeLa cells.10
Mouse studies
Procedures were performed according to protocols approved by the Animal
Care and Use Committee of the Fondazione San Raffaele (IACUC #325)
and communicated to the Ministry of Health and local authorities. Mice
genotyping was performed on DNA from tail biopsies as described.11
Isolation, transduction, and transplantation of murine
hematopoietic cells
Homing assay
Competitive repopulation assay
Lineage⫺ HSPCs were isolated from 4-week-old CD45.2 Twitcher mice
and from CD45.1 wild-type donors. After overnight transduction with
PGK.GFP LV (see above for transduction protocol), cells were washed,
mixed in 1:1 proportion and transplanted intravenously (1.6 ⫻ 105 total
cells/mouse for the low dose group or 3 ⫻ 105 total cells/mouse for the high
dose group) in 2-month-old lethally irradiated CD45.1 recipients. Engraftment was analyzed on peripheral blood by FACS analysis (CD45 missmatch and GFP expression) at 8 and 16 weeks after the transplant.
GALC activity
GALC activity was determined as described.14
Quantitative PCR
Five-week-old mice were killed with CO2, and the bone marrow (BM) was
harvested by flushing the femurs and tibias. Different populations were
isolated and transplanted into 8-day-old lethally irradiated (900 rad) mice:
(1) total BM to be transplanted as a whole (2 ⫻ 106 cells/mouse); (2)
HSPCs, which were purified by lineage negative (lineage⫺) selection using
the Stem Sep Separation kit (StemCell Technologies Inc.) and transplanted
intraperitoneally (1 ⫻ 106 cells/mouse) or intravenously (5 ⫻ 105 cells/
mouse); (3) c-Kit⫹, Lineage⫺, Sca1⫹ (KLS) cells, which were isolated as
c-Kit⫹, Sca1⫹ cells from lineage⫺ isolated HSPCs by Cell Activated
Sorting (Vantage Diva apparatus; Becton Dickinson). 5 ⫻ 104 KLS cells/
mouse were transplanted intravenously; and (4) Sca1⫹ cells were depleted
from whole BM by Sca1⫹ selection kit (MACS; Miltenyi Biotec) and the
negative fraction was collected. Aliquots of 2 ⫻ 107 Sca1⫺ cells/mouse
were transplanted.
All these hematopoietic populations (except for Sca1⫺ cells) were
tracked in vivo by green fluorescent protein (GFP) expression. HSPCs were
transduced with PGK.GFP LV at multiplicity of infection (MOI) 100 in the
absence of serum and with cytokines (100 ng/mL murine stem cell factor
[SCF], 100 ng/mL human FMS-like tyrosine kinase 3 ligand [hFlt3L],
100 ng/mL mouse interleukin 3 [IL-3], 200 ng/mL human IL-6) for
12 hours, as described.12 Untransplanted cells were kept in culture as
described.12 Total BM, HSPCs, and KLS cells were retrieved from GFP
transgenic mice, which were generated as described9 and screened for GFP
expression in peripheral blood mononuclear cells by fluorescence-activated
cell sorting (FACS) analysis before use; only animals showing GFP
expression in at least 90% of the cells were used.
Engraftment was analyzed on peripheral blood and/or bone marrow by
FACS analysis (as percentage of GFP⫹ cells) and/or by quantitative
polymerase chain reaction (PCR) at ⫹120 days after transplantation (and
⫹20, in selected cases) or, in case of earlier death of transplanted animals,
at mouse death.
Isolation and transduction of human HSPCs
Human HSPCs were isolated by positive selection of CD34-expressing
cells (CD34 progenitor cell isolation kit; MACS) from cord blood (CB) or
Genomic DNA was extracted from liquid culture samples with QIAamp
DNA Blood Mini kit (QIAGEN). Quantitative PCR analysis was performed
on at least 50 ng of total genomic DNA as described.13,15 The number of LV
copies per cell (vector copy number [VCN]) was calculated by the
following equation: (ng LV/ng endogenous DNA) ⫻ (n° of LV integrations
in the standard curve).
Colony-forming cell assay
After transduction, HSPCs were washed, counted, and seeded at a density
of 1000 cells/mL (human) or 5000 cells/mL (murine) in semisolid medium
(MethoCult). After 10 days, colonies were scored and counted.
TUNEL assay
HSPCs were washed twice with PBS, stained with TUNEL In Situ Cell
Death Detection kit, TMR red (Roche), and analyzed by confocal
microscopy.
Antiapoptotic treatment
HSPCs, washed after LV transduction, were treated in vitro with insulinlike growth factor 1 (IGF1) 50 ng/mL in RPMI without serum at room
temperature (RT) for 40 minutes.
Immunofluorescence
Cells were seeded on Matrigel-coated coverslips, fixed with PBS 4%
paraformaldehyde, blocked with PBS 1% bovine serum albumin (BSA),
5% goat serum for 1 hour at RT, and incubated with the primary antibody in
PBS 10% goat serum, 0.3% Triton for 2 hours at RT or overnight at 4°C
(Ceramide, Alexis Biochemicals; and Lamp1, Abcam). After washing,
samples were incubated for 1 hour at RT with the conjugated secondary
antibody (Alexa Fluor; Molecular Probes) 1:500 in PBS 0.1% Triton.
Nuclei were labeled with ToProIII (Molecular Probes).
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BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
Sphingolipid analysis
Quantification of sphingolipids was conducted by liquid chromatography,
electrospray tandem mass spectrometry as described.16
Culture and transduction of U937 cells
U937 cells were kept in culture in RPMI medium 10% FBS (Hyclone),
penicillin 100 U/mL, and streptomycin 100 ␮g/mL, L-glutamine 2 mM.
Transduction was performed at MOI 50 and 100 for 12 hours.
Western blot analysis
Western blot analysis was performed as described17 using the following
primary antibodies: anti-Cathepsin D, anti-PAR4, and anti-b-actin (Santa
Cruz Biotechnology), anti-Caspase-9, and anti-Caspase-3 (Cell Signaling
Technology). Immunostaining was performed using the enhanced chemiluminescence (ECL) kit (GE Healthcare, United Kingdom).
Annexin V staining
To detect early apoptotic cells the annexin V–PE apoptosis detection kit
(Becton Dickinson) was used. After staining with specific antibodies for
surface markers, cells were washed with PBS and resuspended in Binding
Buffer at the concentration of 106 cells/mL and labeled following manufacturer’s protocol. 7-Amino-actinomycin D (7-AAD) was also added to the
cells to exclude from the analysis dead cells. Samples were analyzed by
flow cytometry (Canto II) within 1 hour, and results were analyzed by the
Flow-Jo 8.3 software.
Hoechst 33 342 proliferation assay
After staining with specific antibodies for surface markers, cells were
washed with PBS and incubated in the presence of 5 ␮g/mL Hoescht 33 342
for 20 minutes at 37°C in the dark. 7-AAD was then added, and samples
were analyzed using Canto II.
Statistical analysis
Analyses were made by 1-way analysis of variance (ANOVA) for repeated
measurements using Bonferroni’s test for post-hoc analysis and by unpaired
t test (confidence interval 95%).
Results
GALC deficiency affects the compositions of the hematopoietic
stem and progenitor cell compartment
To assess whether GALC deficiency could account for defects in
hematopoiesis, we studied the BM of the Galc natural mutant
Twitcher mouse in the FVB background (FVB/Twi).18 Differently
from what was observed in the thymus, spleen, and liver,19 the BM
cellular content was equal between Galc mutant (⫺/⫺) and
wild-type (⫹/⫹) animals (not shown). However, KLS cells, which
contain HSC activity in mice, were significantly reduced in
frequency and absolute counts in ⫺/⫺ as respect to ⫹/⫹ controls
(Figure 1A); further, within mutant KLS, multipotent progenitors
type 1 (MPP1) and lineage restricted/granulocyte-monocytelymphocyte progenitors (GMLP) were less abundant than in
unaffected controls (Figure 1B), as analyzed by multicolor cytofluorimetry.20,21 The characterization of committed progenitors (Figure
1C) and of more mature cells (Figure 1D-F) revealed a low
frequency of granulocytes and an expansion of orthochromatic
erythroblasts in ⫺/⫺ mice.
Having observed a reduction in the frequency of mutant KLS
cells, and in particular of the more committed fractions within this
GALC AFFECTS HSPC SURVIVAL
1859
population, we wanted to assess whether these cells exhibited an
altered proliferation rate or were more prone to apoptosis compared
with their ⫹/⫹ counterpart. Interestingly, cytofluorimetric analysis
using the Hoechst 33 342 dye, which binds DNA during cells
division, demonstrated that bona fide CD150⫹ CD48⫺ long-term
HSC (LT-HSC, the more immature KLS fraction) retrieved from
Galc⫺/⫺ mice proliferated less than their wild-type counterpart
(Figure 1G). Conversely, no increase in the frequency of annexin
V⫹ cells was observed in any of the ⫺/⫺ fractions analyzed, and
rather a lower incidence of apoptosis in ⫺/⫺ LT-HSC compared
with ⫹/⫹ cells was noticed (Figure 1H). These 2 observations
indicate that the reduced frequency of KLS in the BM of mutant
mice might be due to a below-normal proliferation of early
progenitors, resulting in a defect in the frequency and overall
number of multipotent progenitors.
Interestingly, and in agreement with these findings, when
clonogenic assays were performed on mutant HSPCs retrieved
from the BM of GLD mice, a significantly lower number of
colonies was obtained from Galc⫺/⫺ compared with ⫹/⫹ HSPCs,
both in the case of cytokine prestimulation and of its absence
(Figure 1I), with a normal proportion of erythroid versus myeloid
colonies (not shown). A similar clonogenic impairment was also
detected in human CD34⫹ isolated from GLD patients (Figure 1J).
HSPCs obtained from mice and patients affected by metachromatic
leukodystrophy (MLD), a LSD caused by the deficiency of the
ARSA enzyme that acts upstream GALC in the sulfatide
catabolic pathway, were instead intact in their clonogenic
potential (Figure 1I-J).
GALC deficiency results in HSPC functional impairment
To unravel whether these alterations in the composition of ⫺/⫺
stem and progenitor cells could have functional consequences, we
performed in vivo functional studies (Figure 2A). Galc⫺/⫺ and
⫹/⫹ murine lineage⫺ HSPCs were efficiently transduced with
GFP-encoding LV (PGK.GFP LV; 95% ⫾ 4% GFP⫹ cells) for
donor cell tracking (Figure 2B). The transduced cells were then
transplanted intraperitoneally into heterozygous (⫹/⫺) or Galc⫺/⫺,
8-day-old, lethally irradiated syngeneic recipients. The short-term
homing of the transplanted cells to the BM (within 20 hours from
transplant) was similar in all the transplant pairs (Figure 2C). In the
longer term, however, death due to myeloablation (in between
⫹14 and ⫹21 days posttransplantation, thus before 28 postnatal
days, similar to what observed in irradiation controls; data not
shown) and elevated frequency of engraftment failure (defined as
death at ⱕ 21 days postirradiation, and/or ⱕ 5% donor cells in the
BM at death or at ⫹120 postnatal days) were observed in ⫺/⫺
animals receiving Galc⫺/⫺ HSPCs (Figure 2D-F). Heterozygous
mice transplanted with Galc⫺/⫺ cells survived long-term, but in
the presence of autologous reconstitution (namely in the presence
of ⱕ 5% of GFP⫹ cells in the BM, being the donor cells GFP⫹ at a
frequency equal to 95% ⫾ 4%; Figure 2D-F). Conversely, transplantation of ⫹/⫹ HSPCs into both Galc⫺/⫺ and wild-type recipients
resulted in survival after myeloablation, low engraftment failure
frequency, and good donor chimerism (Figure 2D-F), suggesting
that Galc⫺/⫺ HSPCs are defective in their repopulation ability
compared with Galc⫹/⫹ cells. Interestingly, the lower donor
chimerism observed when wild-type HSPCs were transplanted into
Galc⫺/⫺ recipients as respect to ⫹/⫺ mice suggests that in
Galc⫺/⫺ mice a niche defect could also be present.
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VISIGALLI et al
BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
Figure 1. Phenotype and clonogenic potential of Galcⴚ/ⴚ HSPCs. (A-F) Immunophenotypic characterization of the BM of Galc⫺/⫺ mice. Frequency and absolute cell
number (#) of c-kit⫹ Sca1⫹ cells within lineage⫺ (Lin)⫺ BM cells (KLS; A), and of long-term HSC (LT-HSC), MPP1, MPP2, and GMLP (within the KLS fraction) of ⫺/⫺ and ⫹/⫹
mice (B). Frequency of lymphoid and myeloid progenitors within Lin⫺ cells (C) of wild-type and mutant mice. Lymphoid (D), erythroid (E), and myeloid (F) differentiation in ⫺/⫺
and ⫹/⫹ BM. Each dot represents a pool of 3 mice in panels A through C and a single mouse in panels D through F; means are shown. For details on the gating strategy, please
see supplemental Table 1. (G-H) Frequency of proliferating cells at Hoechst staining (G) and of apoptotic annexin V positive cells (H) within KLS, LT-HSC, MPP1, MPP2, and
GMLP from ⫹/⫹ and ⫺/⫺ mice. Each dot represents a pool of 3 mice; means are shown. (I-J) Number (#) of colonies retrieved from colony-forming cell assays performed with
murine (I) and human (J) HSPCs from wild-type (Galc⫹/⫹ and Arsa⫹/⫹), Galc⫺/⫺ and Arsa ⫺/⫺ mice, and from normal donors’ (nd) and GLD/MLD patients’ BM. Before
plating, murine HSPCs were (w) or were not (w/o) prestimulated with a standard cytokine cocktail for 12 hours.12 n ⱖ 10 (murine) and n ⱖ 3 (human). *P ⬍ .05, **P ⬍ .01 on
1-way ANOVA.
To confirm the functional defect observed in Galc⫺/⫺ HSPCs,
competitive repopulation experiments were performed in a congenic reporter setting. Galc⫺/⫺ HSPCs retrieved from Twitcher
mice (in C57Bl/6 background and expressing the CD45.2 antigen
on cell surface) and HSPCs from wild-type donors carrying the
CD45.1 allele were transduced with the PGK.GFP LV at high
efficiency and then transplanted into lethally irradiated CD45.1
adult recipients by intravenous administration at 1:1 ratio;
2 different cell doses were used (1.6 and 3 ⫻ 105 total cells;
Figure 2G). Frequency of the CD45.1⫹ and CD45.2⫹ cells and
GFP expression within each subset were measured 8 and
16 weeks after the transplantation on peripheral blood mononuclear cells. Interestingly, a significantly lower frequency of
Galc⫺/⫺ CD45.2 cells as respect to Galc⫹/⫹ CD45.1 cells was
observed at both cell doses, with much lower CD45.2 cell
engraftment at the lowest dose (Figure 2H). Of note, the
frequency of GFP⫹ cells within each of the 2 antigen missmatched populations was similar and above 95%, confirming
that lethal myeloablation occurred after irradiation and that the
detected CD45.1 cells were of donor origin (Figure 2I).
GALC deficiency causes a microenvironmental defect that
reduces the repopulating activity of wild-type HSPCs
To further assess the functional integrity of the BM hematopoietic niche of GLD mice, different GFP⫹ hematopoietic populations from Galc⫹/⫹ donors were transplanted into lethally
irradiated 8-day-old Galc⫺/⫺ syngeneic animals (Figure 3A,D).
The different populations to be transplanted were distinguished
according to a progressive enrichment of the stem cell fraction
and to depletion of committed cells (Figure 3A,D). In particular,
the following populations were used: (1) total BM from GFP
transgenic mice (generated by LV transgenesis using the
PGK.GFP LV used for HSPC transduction, and having ⬎ 90%
of their hematopoietic cells expressing GFP); (2) lineage⫺
HSPCs from GFP transgenic mice plus the Sca1⫺ fraction of the
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BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
GALC AFFECTS HSPC SURVIVAL
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Figure 2. In vivo functional characterization of Galcⴚ/ⴚ HSPCs. (A) Experimental scheme for panels D through F. (B) Representative cytofluorimetric dot plots of
GFP-transduced HSPCs and their negative control. (C) Frequency of GFP positive cells in the BM of FVB/Twi Galc⫺/⫺ and ⫹/⫹ recipients 20 hours after the transplantation of
GFP⫹ Galc⫺/⫺ and ⫹/⫹ HSPCs (n ⫽ 4 for each group). (D) Survival curve of Galc⫺/⫺ and ⫹/⫺ recipient mice (rec.) after the intraperitoneal transplantation of Galc ⫹/⫹ or
⫺/⫺ HSPCs. (E-F) Engraftment failure frequency (E; defined as percent of animals showing a survival less than or equal to 21 days postirradiation, and/or less than or equal to
1% donor cells in the BM at death or at 120 postnatal days) and donor chimerism (% of GFP⫹ cell in the BM) at 120 days after transplantation or at mice death (F), measured in
the same experimental groups as in panel D. Average values ⫾ SD are reported. (G) Experimental scheme for competitive transplantation experiments (H-I). (H) Engraftment
of Galc⫹/⫹ CD45.1⫹ and of Galc⫺/⫺ CD45.2⫹ cells in peripheral blood of wild-type CD45.1 mice transplanted with HSPCs measured at 8 and 16 weeks (ws) after the
transplantation (high or the low dose, see text for details; n ⫽ 6 for each group). Average values ⫾ SD are reported. (I) Representative plots from cytofluorimetric analysis
showing engraftment of CD45.1 and CD45.2 cells on peripheral blood of mice receiving competitive transplantation and GFP expression in each of the 2 populations.
BM of wild-type syngeneic mice (GFP negative); (3) HSPCs
from GFP transgenic mice; and (4) KLS from the same GFP
transgenic animals. These populations were administered either
intraperitoneally or intravenously, as indicated in Figure 3A,D.
FVB/Twi mice were also transplanted intravenously to favor
homing of the cells to the BM, whereas Twitcher mice could
only be transplanted intraperitoneally.
Prolonged survival above untreated controls, high chimerism of
donor cells, and low frequency of engraftment failure were
observed in FVB/Twi mice only upon intravenous transplantation
of HSPCs (Figure 3B-C). KLS cells transplanted intravenously and
HSPCs transplanted intraperitoneally gave rise to engraftment
failure at high frequency and to low donor chimerism (with
autologous reconstitution), resulting in early death likely due to
myeloablation in the first case and death around 50 days due to the
disease in the latter (Figure 3B-C). KLS were unable to repopulate
Twitcher mice (not shown), in which sustained donor chimerism
and long-term survival were obtained only upon transplantation of
populations also comprising committed progenitors (Figure 3E-F).
Importantly, all tested Galc⫹/⫹ populations repopulated with
equal efficiency Galc⫹/⫹ (Figure 3B-C for KLS cells and Figure
2D-F for HSPCs) and Arsa⫺/⫺ mice (data not shown, please refer
to our previous studies12,15), indicating that the BM of Galc⫺/⫺
mice is poorly permissive to syngeneic HSPC engraftment.
Physiologic, but not supranormal GALC expression restores
HSPC function
To assess whether restoration of GALC activity in HSPCs by
means of gene transfer could rescue the functional defect of
Galc⫺/⫺ cells, HSPCs from ⫹/⫹ and ⫺/⫺ mice were transduced
with LV encoding the murine Galc cDNA under the control of
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VISIGALLI et al
BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
Figure 3. Functional characterization of the Galcⴚ/ⴚ niche. (A,D) Experimental schemes. (B,E) Survival curves of Galc⫺/⫺ mice (FVB/Twi in panel B and Twi in panel E)
after the intravenous or intraperitoneal transplantation of GFP⫹ Galc⫹/⫹ HSPC or KLS or total BM (TBM) or HSPC plus GFP⫺ Sca1⫺ BM cells, as indicated. KLS intravenous
(WT): KLS transplanted intravenously in wild-type recipients. n ⱖ 3 in each group. (C,F) Engraftment failure frequency and donor chimerism measured in the same
experimental groups as in panels B and E. Average values ⫾ SD are reported. *P ⬍ .05, **P ⬍ .01 on 1-way ANOVA.
either the human GALC promoter (GALC.Galc LV; kindly provided
by Dr. P. Luzi) or the human PGK promoter (PGK.Galc LV; Figure
4A-B). ARSA was chosen as control lysosomal enzyme that does
not affect HSPC function.13 Upon similar efficient transduction
(measured as number of LV copies integrated per cell, VCN),
partial reconstitution of GALC activity was observed in the
culture progeny of ⫺/⫺ HSPCs transduced with GALC.Galc LV
(35% of wild-type values), while enzyme supraphysiologic
expression was measured in the progeny of both ⫺/⫺ and ⫹/⫹
cells transduced with the PGK.Galc LV (up to approximately
2-fold the values of ⫹/⫹ mock transduced cells; Figure 4B).
Interestingly, ⫺/⫺ HSPCs expressing Galc from the GALC
promoter showed restoration of a normal clonogenic potential
compared with mock-transduced ⫹/⫹ and ⫺/⫺ cells (Figure
4C). Conversely, GALC overexpression driven by the PGK
promoter caused a significant impairment of the clonogenic
potential of both Galc⫺/⫺ and ⫹/⫹ cells compared with ARSA
transduced, genotypically identical cells (Figure 4C). The
functional impairment of PGK.Galc transduced ⫺/⫺ and ⫹/⫹
HSPCs was confirmed in vivo upon intraperitoneal transplantation into ⫺/⫺ and ⫹/⫺ FVB/Twi recipients (Figure 4D-G) and
was likely due to the widespread occurrence of apoptosis of the
GALC overexpressing cells (Figure 4H). Comparable results
were obtained upon PGK.Galc transduction and transplantation
of Galc⫺/⫺ HSPCs in another GLD mouse model that,
differently from the knock outs FVB/Twi and Twitcher mice, is
characterized by residual GALC activity (Trs mice,22 and as
reported in B.G., I.V., H. Hiramatsu, E. Lechman, S.U., A.
Giustacchini, G. Schira, M. Amendola, S.M., A.O., J. E. Dick,
L.N., and A.B., Identification of HSC-specific miRNAs enables
tight regulation of gene expression and effective gene therapy of
globoid leukodystrophy, manuscript submitted). Conversely,
moderate enzyme expression corrected the functional deficit of
mutant HSPCs also in vivo. Indeed, mutant HSPCs transduced
with the GALC.Galc LV efficiently engrafted in the BM of ⫹/⫺
FVB/Twi mice (Figure 4D-G). Thus, while GALC expression
driven by its own promoter restores a normal function of mutant
HSPCs, enzyme supraphysiologic expression further worsens
the functional impairment of ⫺/⫺ HSPCs, causes a functional
impairment of normal cells and apoptotic cell death of both cell
types. Similar findings were also obtained on CD34⫹ HSPCs
obtained from GLD BM and normal donors’ CB (Figure 4I and
B.G., I.V., et al, manuscript submitted).
IGF1 rescues GALC overexpressing HSPCs from apoptosis
and functional impairment
Several studies have implicated IGF1 in preventing cell death due
to the activation of the phosphatidylinositol-3 kinase (PI3K)/Akt
signaling pathway,23 which is also required for the survival of
hematopoietic cells.24 Cer acts on this same pathway by inhibiting
the phosphorylation of Akt and Erk1/Erk2.25,26 After transduction
with PGK.Galc or PGK.ARSA LV, Galc⫺/⫺ murine HSPCs and
CB CD34⫹ cells were briefly (40 minutes) incubated with IGF1.
The treatment rescued PGK.Galc-transduced HSPCs from toxicity;
a significant increase in the number of the colonies (Figure 4C) and
protection of GALC overexpressing cells from apoptosis (Figure
4H-I) were observed. When Galc⫺/⫺ HSPCs transduced with
PGK.Galc and treated with IGF1 were transplanted into irradiated
Galc⫹/⫺ and ⫺/⫺ mice, they were able to prevent recipient
animals from early death and to engraft at sustained levels (Figure
3D-G), confirming that apoptotic cell death of GALC overexpressing HSPCs was responsible for the observed functional impairment. In the long-term, however, transduced cells were no longer
detectable in the BM of the recipients, which died due to GLD
manifestations (⫺/⫺ mice) or survived long-term due to reconstitution by donor nontransduced or autologous cells (⫹/⫺ mice;
Figure 4E-G), because of a likely exhaustion of the effect of the
brief in vitro treatment with IGF1, which was thus unable to protect
the transduced HSPCs from GALC toxicity long term.
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BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
GALC AFFECTS HSPC SURVIVAL
1863
Figure 4. Characterization of the diverse phenotypes associated to alterations of GALC expression in murine and human HSPCs. (A,D) Experimental schemes.
(B) GALC activity (measured as fold to mock-transduced Galc⫹/⫹ cells) and LV copy number (VCN) measured on the in vitro progeny of murine Galc⫺/⫺ and ⫹/⫹ HSPCs
transduced with the indicated LV and exposed or not to IGF1 after transduction. n ⱖ 3 in each group. (C) Number (#) of colonies retrieved from colony-forming cell assays
performed with murine HSPCs obtained from Galc⫺/⫺ and ⫹/⫹ mice after transduction with the specified LV and exposed to IGF1 after transduction, if indicated. n ⱖ 3 in each
group. (E) Survival curves of FVB/Twi Galc⫺/⫺ and ⫹/⫺ mice after the intraperitoneal transplantation of Galc⫺/⫺ HSPCs transduced with the indicated vectors and exposed to
IGF1 after transduction, if indicated. n ⱖ 3 in each group. (F-G) Engraftment failure frequency (F) and transduced cell engraftment (VCN in the BM; G) at 20 and at 120 days
after transplantation or at death as in (*), measured in the same experimental groups as in panel E. (§) Similar results were obtained with ⫹/⫹HSPCs. (H-I) TUNEL assay
performed on murine (H) and human (I) HSPCs obtained from Galc⫺/⫺ and ⫹/⫹ mice and from normal donors (nd) CB and GLD patients’ BM, 2 and 5 days (d) after
transduction with the specified LV and exposed to IGF1 after transduction, if indicated. Percentage of TUNEL⫹ nuclei over the total number of nucleated cells is reported
(ⱖ 8 fields and ⱖ 100 cells were counted per condition). n ⱖ 5 individual experiments per condition, average values ⫾ SD are reported. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001 on
1-way ANOVA.
Functional impairment is associated to variations in the
intracellular content of bioactive GALC products
An intracellular sphingolipid profile was then performed on control
vector and PGK.Galc-transduced murine HSPCs before and at
different intervals from gene transfer. Sphingomyelin (SM), Cer,
Sph, S1P, Psy, and sulfatides (Sulf) were detected in ⫹/⫹ and ⫺/⫺
cells (Figure 5A). SM was the most abundant sphingolipid, and the
C:16 fractions of SM and Cer were the most represented among the
measured isoforms. Gal-Cer was poorly detectable, being in the
range of 0 to 2 pmol/106 cells.
At the beginning of the culture, the intracellular content of Cer
and Sph was significantly lower (approximately 60%) in Galc⫺/⫺
as respect to ⫹/⫹ HSPCs, suggesting that GALC-dependent
metabolism could represent one of the pathways regulating intracellular levels of these sphingolipids in HSPCs. Moreover, a higher
Psy intracellular content was observed in ⫺/⫺ HSPCs, despite not
reaching statistical significance.
Two to 7 days after PGK.Galc transduction, we detected a
progressive accumulation of Cer, Sph, and of S1P both in ⫺/⫺ and
⫹/⫹ HSPCs, compared with basal levels and to GFP controls (up
to 8- and 2-fold increase in respect to the values measured in
freshly isolated ⫺/⫺ cells and GFP control cells at 7 days after
transduction, respectively, in the case of C:16 Cer; Figure 5B and
data not shown for ⫹/⫹ cells). Further, Psy showed a tendency to
decrease upon GALC gene transfer (not shown). Immunofluorescence confirmed the presence of Cer in hematopoietic cells (Figure
5C and data not shown). In GALC transduced cells, Cer signal
colocalized more abundantly with the lysosomal marker Lamp1,
compared with cells transduced with a control vector, suggesting
that the intracellular Cer increase could be due to an increase of the
lysosomally generated Cer.
Interestingly, when murine and human HSPCs were transduced
with a bidirectional LV,9 which allowed the simultaneous expression of GALC (Figure 4B) and of the Cer-degrading enzyme acid
ceramidase (GALC&AC LV), rescue from GALC toxicity was
seen (Figure 4C,H-I). The progressive reduction of TUNEL⫹ cells
by 2 to 5 days after gene transfer suggests an active and progressive
degradation of accumulated Cer by acid ceramidase (Figure 4H-I).
Importantly, when Galc⫺/⫺ HSPCs transduced with the
GALC&AC LV were transplanted in lethally irradiated ⫹/⫺ and
⫺/⫺ recipients, mice were rescued from early lethality in the
presence of transduced cell engraftment (Figure 4D-G).
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1864
VISIGALLI et al
BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
Figure 5. Sphingolipid profile of murine HSPCs. (A) Total sphingolipid content (pmol/106 cells) of Galc⫺/⫺ and ⫹/⫹ murine HSPCs. Each dot represents an individual
sample; means are shown. (B) Intracellular content of C:16 Cer (left panel), Sph (middle panel), and S1P (right panel), in GALC or GFP LV transduced ⫺/⫺ HSPCs at 12 to 24
hours (h), 2 and 7 days (d) after gene transfer (n ⱖ 3). *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001 on 1-way ANOVA. (C) Representative images obtained after immunofluorescence
staining for ceramide and for the lysosomal marker Lamp1 performed on human monocytes (U937 cells) transduced with GALC and control DeltaNGFR (DNGFR) LV 2 and 7
days after transduction. Magnification 100⫻ in the 2 top rows and 200⫻ in the bottom row.
Discussion
Here we describe a defect in the frequency, proliferation, clonogenic potential, and engraftment capability of early hematopoietic
progenitors retrieved from Galc mutant mice and a similar defect in
the clonogenic ability of HSPCs from GLD patients. This defect
was rescued by low-level reconstitution of GALC activity, while
unregulated, supraphysiologic GALC expression rather worsened
the preexisting defect and induced apoptotic death also of wildtype HSPCs. The 2 dysfunctional phenotypes were associated to
alterations of the intracellular content of the bioactive sphingolipid
Cer; functionally defective Galc⫺/⫺ cells had a lower Cer content
compared with normal cells, whereas enzyme overexpressing cells
accumulated Cer, which localized within the lysosomal compartment. A similar pattern applies also to Sph. The hydrolysis of Cer
upon coexpression, together with GALC, of acid ceramidase
rescued HSPC from apoptosis and functional impairment, confirming the key role of this sphingolipid in affecting HSPC function and
survival. In addition, we here also show a microenvironmental
defect of the hematopoietic niche of Galc⫺/⫺ animals that reduces
the repopulating activity of wild-type HSPCs.
LSDs are genetically inherited diseases characterized by the
accumulation of disease-specific metabolic intermediates within
the lysosome. The importance of the lysosomal compartment to
normal cell function is demonstrated by the various pathologies
that arise in LSDs. These disorders are invariably fatal, and many
display profound neurologic impairment. Recent studies have
revealed that LSDs are also characterized by irregularities in the
function of the immune system.27 Further, in some LSDs the
practice of allogeneic HSC transplantation (HCT) revealed a high
frequency of mixed donor chimerism and of engraftment failure.28
A recent report suggests that lymphomonocytes from GLD patients
could have a basal proinflammatory pattern likely related to Psy
accumulation,29 but no specific abnormalities of HSC have been
investigated or described thus far in GLD patients. Moreover, the
limited experience in HCT in GLD patients has not revealed low
efficiency of donor cell engraftment.30
Most of the mutations of the human GALC gene are responsible
for a reduction of enzyme activity but do not abrogate enzyme
production. The residual, low enzyme activity present in most of
the patients but not in Twitcher and FVB/Twi mice, which are
natural knockouts, could be responsible for the lack of an overt
phenotype in the patients. This could be consistent with the low
level of GALC protein detected in tissues from normal subjects and
the very strict therapeutic window for GALC activity shown by our
gene transfer studies in HSPCs. Very tiny amounts of GALC are
likely required and sufficient for the maintenance of a healthy
hematopoietic niche. Otherwise, the very short life span and the
neurologic symptoms, which prevail in the patients, could have
masked this phenotype.
Our mass spectrometry data obtained in ⫺/⫺ cells, together
with the functional studies using IGF1 and acid ceramidase
indicate that Cer may play a central role in the events consequent to
alterations of GALC activity within HSPCs and, possibly, their
niche. Recently, a novel role of Cer and other sphingolipids in
regulating cell survival, senescence, and apoptosis has been
demonstrated.4 Cer can be considered a metabolic hub because it
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BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
occupies a central position in sphingolipid biosynthesis and
catabolism. Interestingly, our data suggest that GALC, beside other
relevant pathways,31 may contribute to the regulation of the
intracellular levels of lysosomally generated Cer in HSPCs. Similar
variations of intracellular levels of Sph, which appears to act in a
similar fashion as Cer, occurred. On the contrary, S1P, which
increases in the transduced HSPCs but remains at very low
concentration, appears to have opposite effects as Cer in many of
the pathways in which it is involved, particularly in those relating
to cell growth and survival. It has been suggested that the balance
between survival and death in many cell types may be affected by
the equilibrium between intracellular levels of each of these
interconvertible sphingolipids. Enzymes that either produce or
degrade the sphingolipids control this equilibrium. Thus, our
findings suggest that an alteration of the intracellular content of Cer
and, possibly, of other bioactive sphingolipids in HSPCs following
either abrogation of GALC activity (because of a severe mutation
in the gene) or gene transfer and supraphysiologic GALC expression, may substantially affect this balance. Indeed, the increase in
Cer and Sph, despite being modest when considering levels
detected in other tissues or cell types, might exceed intracellular
antiapoptotic signals, like that of S1P, thus triggering apoptosis of
HSPCs. On the contrary a reduction of Cer and Sph content in
HSPC due to GALC deficiency could affect the survival and
function of these cells and the tropism of the hematopoietic niche.
The downstream molecular pathways activated by the imbalance of
Cer, Sph, and S1P relative ratios in HSPCs are not yet completely
understood (supplemental Figure 1).
Interestingly, differentiated hematopoietic cells are not affected
by GALC overexpression (B.G., I.V., H. Hiramatsu, E. Lechman,
S.U., A. Giustacchini, G. Schira, M. Amendola, S.M., A.O., J. E.
Dick, L.N., and A.B., Identification of HSC-specific miRNAs enables
tight regulation of gene expression and effective gene therapy of
globoid leukodystrophy, manuscript submitted), suggesting a unique
sensitivity of HSPCs to GALC- and sphingolipid-mediated control
of cell survival. Further studies will elucidate the actual mechanisms responsible for this peculiar sensitivity.
The defect in the mutant hematopoietic stem cell niche here
described could be interpreted in light of the previous reports in the
Twitcher mouse thymus2 and in CGT mutant mice3 as a results of a
defective sympathetic innervation of the niche. However, the role
of the described spingolipid imbalances in contributing to the niche
defect or of the altered niche in aggravating the cell-intrinsic defect
GALC AFFECTS HSPC SURVIVAL
1865
of mutant HSPCs remains to be determined. Interestingly, recent
literature has demonstrated that S1P regulates the SDF-1/CXCR4
axis and, consequently, the interaction of the HSPCs with their
niche as well as critical events like HSPC chemotaxis, in vivo
homing, and recirculation.32,33 Moreover, S1P mobilizes osteoclast
precursors and regulates bone homeostasis.34,35 Interestingly, since
in our experimental model GALC, besides Cer, is also critically
affecting S1P concentration in HSPCs, we could hypothesize that
GALC deficiency and overexpression affects the SDF-1/CXCR4
axis and/or the trophism of the endosteal niche.
Overall, our findings demonstrate that both GALC deficiency
and forced GALC expression affect HSPC survival by altering a
delicate intracellular bioactive sphingolipid balance, showing a
possible novel regulatory role for this enzyme.
Acknowledgments
We are indebted to A. Merrill for tandem mass spectrometry
analyses; J. Medin for the acid ceramidase cDNA; P. Luzi and D.
Wenger for the GALC promoter sequence; S. Marchesini, I. di
Girolamo, and R. Tiribuzi for biochemical studies; and A. Rovelli
for GLD patients’ BM.
This research was supported by grants from the Italian Telethon
to A.B. and L.N., the European Leukodystrophy Association and
EU (FP7 LEUKOTREAT 241622) to A.B., the Italian Ministry of
Health (PS NEURO ex 56-05-16) and the Cariplo Foundation
(Cariplo NOBEL) to L.N., and the National Institutes of Health
(GM069338) to A. Merrill.
Authorship
Contribution: I.V. performed research and contributed to experiment design; S.U., S.M., H.P., M.C., and A.O. performed research
and analyzed the data; B.G. provided protocols; L.S.S. provided
reagents; L.N. interpreted the data; and A.B. designed experiments,
interpreted the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Alessandra Biffi, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Via
Olgettina 58, Milan, Italy; e-mail: [email protected].
References
1. Suzuki K. Twenty five years of the “psychosine
hypothesis”: a personal perspective of its history
and present status. Neurochem Res. 1998;23(3):
251-259.
2. Galbiati F, Basso V, Cantuti L, et al. Autonomic
denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of
Krabbe disease. J Neurosci. 2007;27(50):1373013738.
3. Katayama Y, Battista M, Kao WM, et al. Signals
from the sympathetic nervous system regulate
hematopoietic stem cell egress from bone marrow. Cell. 2006;124(2):407-421.
4. Hannun YA, Obeid LM. Principles of bioactive
lipid signalling: lessons from sphingolipids. Nat
Rev Mol Cell Biol. 2008;9(2):139-150.
5. Chen YQ, Wenger DA. Galactocerebrosidase
from human urine: purification and partial characterization. Biochim Biophys Acta. 1993;1170(1):
53-61.
6. Chen YQ, Rafi MA, de Gala G, Wenger DA. Cloning and expression of cDNA encoding human galactocerebrosidase, the enzyme deficient in
globoid cell leukodystrophy. Hum Mol Genet.
1993;2(11):1841-1845.
11. Sakai N, Inui K, Tatsumi N, et al. Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher
mouse, a model of Krabbe’s disease. J Neurochem. 1996;66(3):1118-1124.
7. Luzi P, Victoria T, Rafi MA, Wenger DA. Analysis
of the 5⬘ flanking region of the human galactocerebrosidase (GALC) gene. Biochem Mol Med.
1997;62(2):159-164.
12. Biffi A, De Palma M, Quattrini A, et al. Correction
of metachromatic leukodystrophy in the mouse
model by transplantation of genetically modified
hematopoietic stem cells. J. Clin. Invest. 2004;
113(8):1118-1129.
8. Luzi P, Rafi MA, Wenger D. Structure and organization of the human galactocerebrosidase
(GALC) gene. Genomics. 1995;26:407-409.
9. Amendola M, Venneri MA, Biffi A, Vigna E,
Naldini L. Coordinate dual-gene transgenesis
by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol. 2004;23(1):
108-116.
10. Follenzi A, Naldini L. HIV-based vectors. Preparation and use. Methods Mol. Med. 2002;69:259274.
13. Capotondo A, Cesani M, Pepe S, et al. Safety of
arylsulfatase A overexpression for gene therapy
of metachromatic leukodystrophy. Hum Gene
Ther. 2007;18:821-836.
14. Martino S, Tiribuzi R, Tortori A, et al. Specific determination of ␤-galactocerebrosidase activity via
competitive inhibition of ␤-galactosidase. Clin
Chem. 2009;55(3):541-548.
15. Biffi A, Capotondo A, Fasano S, et al. Gene
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1866
16.
17.
18.
19.
20.
21.
VISIGALLI et al
therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice.
J. Clin. Invest. 2006;116(1):3070-3082.
Merrill AHJ, Sullards MC, Allegood JC, Kelly S,
Wang E. Sphingolipidomics: high-throughput,
structure-specific, and quantitative analysis of
sphingolipids by liquid chromatography tandem
mass spectrometry. Methods. 2005;36(2):207224.
Martino S, Emiliani C, Orlacchio A, Hosseini R,
Stirling JL. ␤-N- acetylhexosaminidases A and
S have similar sub-cellular distributions in HL-60
cells. Biochim Biophys Acta. 1995;1243:489-495.
Visigalli I, Moresco RM, Belloli S, et al. Monitoring
disease evolution and treatment response in lysosomal disorders by the peripheral benzodiazepine receptor ligand PK11195. Neurobiol Dis.
2009;34(1):51-62.
Plati T, Visigalli I, Capotondo A, et al. Development and maturation of invariant natural killer
T cells in the presence of lysosomal engulfment.
Eur J Immunol. 2009;39(10):2748-2754.
Yilmaz OH, Kiel MJ, Morrison SJ. SLAM family
markers are conserved among hematopoietic
stem cells from old and reconstituted mice and
markedly increase their purity. Blood. 2006;107(3):
924-930.
Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH,
Terhorst C, Morrison SJ. SLAM family receptors
distinguish hematopoietic stem and progenitor
cells and reveal endothelial niches for stem cells.
Cell. 2005;121(7):1109-1121.
BLOOD, 16 SEPTEMBER 2010 䡠 VOLUME 116, NUMBER 11
22. Luzi P, Rafi M, Zaka M, Curtis M, Vanier MT,
Wenger D. Generation of a mouse with low galactocerebrosidase activity by gene targeting: a new
model of globoid cell leukodystrophy (Krabbe disease). Mol Genetics and Metab. 2001;73:211223.
23. Laviola L, Natalicchio A, Giorgino F. The IGF-I
signaling pathway. Curr Pharm Des. 2007;13(7):
663-669.
24. Luzi P, Rafi MA, Zaka M, et al. Biochemical and
pathological evaluation of long-lived mice with
globoid cell leukodystrophy after bone marrow
transplantation. Mol Genet Metab. 2005;86(1-2):
150-159.
25. Miyaji M, Jin ZX, Yamaoka S, et al. Role of membrane sphingomyelin and ceramide in platform
formation for Fas-mediated apoptosis. J Exp
Med. 2005;202(2):249-259.
26. Stoica BA, Movsesyan VA, Knoblach SM, Faden
AI. Ceramide induces neuronal apoptosis through
mitogen-activated protein kinases and causes
release of multiple mitochondrial proteins. Mol
Cell Neurosci. 2005;29(3):355-371.
27. Castaneda JA, Lim MJ, Cooper JD, Pearce DA.
Immune system irregularities in lysosomal storage disorders. Acta Neuropathol. 2008;115(2):
159-174.
28. Rovelli AM, Steward CG. Hematopoietic cell
transplantation activity in Europe for inherited
metabolic diseases: open issues and future directions. Bone Marrow Transplant. 2005;35(S1):2326.
29. Formichi P, Radi E, Battisti C, et al. Psychosineinduced apoptosis and cytokine activation in immune peripheral cells of Krabbe patients. J Cell
Physiol. 2007;212(3):737-743.
30. Escolar ML, Poe MD, Provenzale JM, et al.
Transplantation of umbilical-cord blood in babies
with infantile Krabbe’s disease. N Engl J Med.
2005;352(20):2069-2081.
31. Bartke N, Hannun YA. Bioactive sphingolipids:
metabolism and function. J Lipid Res. 2009;
50(Suppl):S91-S96.
32. Ryser MF, Ugarte F, Lehmann R, Bornhäuser M,
Brenner S. S1P(1) overexpression stimulates
S1P-dependent chemotaxis of human CD34⫹
hematopoietic progenitor cells but strongly inhibits SDF-1/CXCR4-dependent migration and in
vivo homing. Mol Immunol. 2008;46(1):166-171.
33. Massberg S, Schaerli P, Knezevic-Maramica I, et
al. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and
peripheral tissues. Cell. 2007;131(5):994-1008.
34. Ishii M, Egen JG, Klauschen F, et al. Sphingosine-1phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature. 2009;458(7237):
524-528.
35. Ryu J, Kim HJ, Chang EJ, Huang H, Banno Y,
Kim HH. Sphingosine 1-phosphate as a regulator
of osteoclast differentiation and osteoclastosteoblast coupling. EMBO J. 2006;25(24):58405851.
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2010 116: 1857-1866
doi:10.1182/blood-2009-12-256461 originally published
online May 28, 2010
The galactocerebrosidase enzyme contributes to the maintenance of a
functional hematopoietic stem cell niche
Ilaria Visigalli, Silvia Ungari, Sabata Martino, Hyejung Park, Martina Cesani, Bernhard Gentner, Lucia
Sergi Sergi, Aldo Orlacchio, Luigi Naldini and Alessandra Biffi
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