Induced pluripotent stem cell model recapitulates pathologic hallmarks of Gaucher disease.
ABSTRACT Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.
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ABSTRACT: More than 500 rare genetic bone disorders have been described, but for many of them only limited treatment options are available. Challenges for studying these bone diseases come from a lack of suitable animal models and unavailability of skeletal tissues for studies. Effectors for skeletal abnormalities of bone disorders may be abnormal bone formation directed by osteoblasts or anomalous bone resorption by osteoclasts, or both. Patient-specific induced pluripotent stem cells (iPSCs) can be generated from somatic cells of various tissue sources and in theory can be differentiated into any desired cell type. However, successful differentiation of hiPSCs into functional bone cells is still a challenge. Our group focuses on the use of human iPSCs (hiPSCs) to identify osteoclast defects in craniometaphyseal dysplasia. In this review, we describe the impact of stem cell technology on research for better treatment of such disorders, the generation of hiPSCs from patients with rare genetic bone disorders and current protocols for differentiating hiPSCs into osteoclasts.Journal of clinical medicine. 12/2014; 3(4):1490-1510.
Chapter: Rare cancers[Show abstract] [Hide abstract]
ABSTRACT: According to IARC a figures "rare and less common" cancers comprise more than a third of all cancer diagnoses as a group. However, advances in molecular biology have resulted in novel ways to classify cancers based upon genetic alterations and not just anatomical location, and this revised classification is at the heart of any move toward more personalised healthcare. It is now increasingly accepted that cancer should be thought of as many hundreds of more rare subtypes, each of which will have specific therapeutic options. We have selected colorectal carcinoma to illustrate the concept that each cancer is "rare", and demonstrate why this is important for delivering on the concept of Predictive, Preventive and Personalised Medicine (PPPM) for cancer in terms of prediction of who will get the disease, how it will behave and how to prevent it.Rare diseases: Integrative PPPM approach as the medicine of the future, Edited by Ozguc, 01/2015: pages 109-130; Springer., ISBN: 2211-3495
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ABSTRACT: Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.Science translational medicine 06/2014; 6(240):240ra73. · 14.41 Impact Factor
Induced pluripotent stem cell model recapitulates
pathologic hallmarks of Gaucher disease
Leelamma M. Panickera, Diana Millera, Tea Soon Parkb,c, Brijesh Patela, Judi L. Azevedoa, Ola Awada, M. Athar Masoodd,
Timothy D. Veenstrad, Ehud Goldine, Barbara K. Stubblefielde, Nahid Tayebie, Swamy K. Polumuria, Stefanie N. Vogela,
Ellen Sidranskye, Elias T. Zambidisb,c, and Ricardo A. Feldmana,1
aDepartment of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201;bInstitute for Cell Engineering andcSidney
Kimmel Comprehensive Cancer Center at The Johns Hopkins University School of Medicine, Baltimore, MD 21205,dLaboratory of Proteomics and Analytical
Technologies, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702; andeMedical Genetics Branch, National Human
Genome Research Institute, National Institutes of Health, Bethesda, MD 20892
Edited by George Q. Daley, Children’s Hospital Boston, Boston, MA, and accepted by the Editorial Board September 13, 2012 (received for review May
Gaucher disease (GD) is an autosomal recessive disorder caused by
mutations in the acid β-glucocerebrosidase gene. To model GD, we
generated human induced pluripotent stem cells (hiPSC), by reprog-
ramming skin fibroblasts from patients with type 1 (N370S/N370S),
type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency
was demonstrated by the ability of GD hiPSC to differentiate to all
three germ layers and to form teratomas in vivo. GD hiPSC differ-
entiated efficiently to the cell types most affected in GD, i.e., macro-
phages and neuronal cells. GD hiPSC-macrophages expressed
macrophage-specific markers, were phagocytic, and were capable
of releasing inflammatory mediators in response to LPS. Moreover,
GD hiPSC-macrophages recapitulated the phenotypic hallmarks of
the disease. They exhibited low glucocerebrosidase (GC) enzymatic
activity and accumulated sphingolipids, and their lysosomal func-
tions were severely compromised. GD hiPSC-macrophages had a de-
fect in their ability to clear phagocytosed RBC, a phenotype of
tissue-infiltrating GD macrophages. The kinetics of RBC clearance
by types 1, 2, and 3 GD hiPSC-macrophages correlated with the
severity of the mutations. Incubation with recombinant GC com-
pletely reversed the delay in RBC clearance from all three types of
GD hiPSC-macrophages, indicating that their functional defects
were indeed caused by GC deficiency. However, treatment of in-
duced macrophages with the chaperone isofagomine restored
phagocytosed RBC clearance only partially, regardless of genotype.
These findings are consistent with the known clinical efficacies of
recombinant GC and isofagomine. We conclude that cell types
derived from GD hiPSC can effectively recapitulate pathologic hall-
marks of the disease.
Gaucher model|Gaucher macrophages|lipid storage disease|
carrier frequency is 1 in 18 and disease incidence is 1 in 1,000,
compared with 1 in 50,000 in the general population (1). GD is
caused by mutations in the acid β-glucocerebrosidase (GBA) gene.
Type 1 GD is the most common and mildest form of the disease
and affects primarily reticuloendothelial cells. The reduced glu-
cocerebrosidase (GC) activity in phagocytic cells results in lyso-
somal accumulation of glucosylceramide (GlcCer) and other
sphingolipids, derived mostly from the breakdown of ingested red
and white blood cell membranes (2, 3). Patients affected with type
1 GD exhibit hepatosplenomegaly, hematologic abnormalities,
and bone disease (1, 4). Type 2 GD is an acute form of the disease
and is usually fatal before 2 y of age. Type 3 GD tends to have
a later onset and is subacute. In patients who have type 2 and
3 GD, in addition to hematologic and visceral manifestations,
there is brain involvement of unclear etiology. Patients with GD
present with heterogeneous symptoms, even among patients with
the same GBA mutations. This heterogeneity suggests that genetic
background and environmental factors may also influence the
severity of the disease (5).
aucher disease (GD) is the most frequently inherited lipid
storage disease; it primarily affects Ashkenazi Jews, where
One obstacle to modeling GD is that the disease-affected cell
types are not easily available. Human macrophages can be
obtained from peripheral blood, but they are difficult to expand in
culture, and patient-derived neuronal cell types are even more
difficult to procure. GD fibroblasts have been widely used for
studying the disease and for therapeutic development, but these
cells are nonphagocytic and do not release the inflammatory
mediators and hydrolases believed to play a role in GD pathology.
Additionally, fibroblasts are of limited utility for studying the un-
derlying causes of neuronopathic GD. These limitations may be
overcome by reprogramming patient-derived cells into human
induced pluripotent stems cells (hiPSC), as first shown by
Yamanaka and coworkers (6, 7). hiPSC have been derived from
patients affected by a variety of diseases (8–11), including long QT
syndrome (12, 13), familial dysautonomia (14), and Alzheimer’s
disease (15), and important aspects of the disease phenotype have
been recapitulated in the relevant hiPSC-derived cell types (16).
In this study we report the development of hiPSC derived from
patients harboring the most frequent mutations associated with
development of types 1, 2, and 3 GD. GD hiPSC were differen-
tiated to macrophages and neuronal cells that were found to
accumulate sphingolipids in a pathologic manner. In mutant
macrophages, GC deficiency resulted in ineffective clearance of
phagocytosed RBC, which is a classic hallmark of the disease (17,
18). Moreover, the extent of the functional defect exhibited by
types 1, 2, and 3 GD hiPSC-macrophages in vitro reflected the
severity of the mutation. Our results suggest that this hiPSC model
recapitulates the phenotypic and pathological variants of the dis-
ease, and can be a valuable tool for understanding molecular mecha-
nisms and developing therapeutic approaches for GD.
Generation of GD hiPSC. GD fibroblasts from patients with types 1,
2, and 3 GD were reprogrammed by expression of SOX2, OCT4,
KLF4, and MYC after infection with the STEMCCA vector, and
initial hiPSC colony selection was based on morphologic re-
semblance to human embryonic stem cell (hESC) colonies (Fig.
S1A). As shown in Fig. 1A, L444P/RecNciI GD hiPSC expressed
typical pluripotency surface markers, including SSEA-3, SSEA-4,
TRA-1–60, and TRA-1–81. They also expressed undifferentiated
Author contributions: L.M.P., D.M., T.S.P., J.L.A., O.A., and R.A.F. designed research;
L.M.P., D.M., T.S.P., B.P., J.L.A., O.A., M.A.M., T.D.V., E.G., N.T., and R.A.F. performed
research; T.S.P., M.A.M., T.D.V., E.G., B.K.S., S.K.P., S.N.V., E.S., and E.T.Z. contributed
new reagents/analytic tools; L.M.P., D.M., T.S.P., B.P., J.L.A., O.A., M.A.M., T.D.V., E.G.,
N.T., S.N.V., E.T.Z., and R.A.F. analyzed data; and L.M.P. and R.A.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. G.Q.D. is a guest editor invited by the
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1207889109 PNAS Early Edition
| 1 of 6
ES cell markers such as NANOG, SOX2, and OCT4 but did not
express SSEA-1, a marker for differentiation in human cells.
Marker analysis was done with five independently derived GD
hiPSC lines, all with similar results. Quantitative analysis of marker
expression by flow cytometry confirmed that the majority of GD
hiPSC expressed these pluripotency markers (Fig. S1B). Similar
analysis of independently derived colonies of N370S/N370S (type
1) and L444P/L444P (type 3) GD hiPSC showed that they all
expressed pluripotency and undifferentiated ES cell markers (see
Fig. S4 A and B). Karyotypic analysis of GD hiPSC lines in-
dicated a normal complement of chromosomes (Fig. S2A).
Pluripotency of GD hiPSC. Pluripotency of GD hiPSC was demon-
strated through their ability to give rise to all three germ layers in
vitro and in vivo. After 15 d in differentiation culture, GD em-
bryoid bodies (EBs) from type 2 L444P/RecNciI hiPSC stained
positive for the ectodermal markers neuronal-specific tubulin
(Tuj1) and microtubule-associated protein 2 (MAP2) (Fig. S2B).
These EBs also were positive for the mesodermal marker bra-
chyury and the endodermal marker GATA4. To assay pluri-
potency in vivo, type 2 GD hiPSC were injected s.c. into NOG/
SCID mice. Teratomas arose within 6–8 wk of injection from all
three GD hiPSC lines tested (Fig. 1 B, a). Histopathological
analysis showed the presence of multiple cell lineages within the
benign tumors, representative of all three germ layers, including
glandular, intestinal, neuronal, bone, and cartilage structures from
the type 2 hiPSC (Fig. 1 B, b–f). The type 1 (N370S/N370S) and
type 3 (L444P/L444P) hiPSC also were pluripotent, as assessed in
vitro and by teratoma formation in NOG/SCID mice in vivo (see
Fig. S4 C and D).
Directed Differentiation of GD hiPSC to Monocyte/Macrophages. GD
hiPSC cultured in suspension as EBs for a week differentiated into
hematopoietic cells that stained positive for CD11b and CD14
(Fig. S3B). From around day 15 onwards, round monocyte-like
cells arose from the middle of the flattened EBs and floated in the
culture medium. Fig. S3A shows different stages of an EB culture
that led to monocyte production. Monocytes harvested from the
culture supernatant showed a single uniform population, and
more than 95% of both GD hiPSC- and control hiPSC-monocytes
expressed CD14 (Fig. 2A and Fig. S2C, respectively). Under
optimal conditions, monocyte-producing factories yielded more
than one million cells per four or five EBs every 4–5 d. The
cultures produced monocytes for more than 3 mo, but yields
diminished with time.
To generate GD macrophages, GD hiPSC-monocytes were
plated onto adherent plates in the presence of M-CSF. After 3 d,
May–Grünwald–Giemsa staining showed the presence of cells
with the typical appearance of macrophages with round or spread-
out morphologies (Fig. 2 C, a–c). These cells expressed typical
markers of macrophage differentiation including CD14, CD163,
and CD68 (Fig. 2 B, a–c). Similar results were obtained by di-
rected differentiation of control hiPSC (Figs. S2 D, a–c and S3C)
and H9/hESC (Fig. S3D), as well as N370S/N370S (Fig. S4E) and
L444P/L444P hiPSC (Fig. S4F).
GD hiPSC-Macrophages Phagocyte Opsonized RBC. Tissue macro-
phages have multiple functions, including the removal of aged
and damaged red and white blood cells from circulation. To
determine whether GD macrophages were able to carry out
phagocytosis of RBC, L444P/RecNciI type 2 GD macrophages
were incubated with opsonized RBC for 2 h at 37 °C. As shown
in Fig. 2 D, a and b (live-cell images) and Fig. 2 D, d and e (May–
Grünwald–Giemsa staining), the GD hiPSC-macrophages had
high phagocytic activity, and the majority of cells were able to
ingest 15–50 RBC. This activity also was seen in macrophages
derived from two other type 2 GD hiPSC lines we tested. RBC
ingestion by GD hiPSC-macrophages was similar to that in
control hiPSC- and hESC-macrophages (Fig. S3 E, a and F, a),
suggesting that GC deficiency did not alter the erythrophagocytic
activity of the mutant macrophages. Type 1 N370S/N370S and
type 3 L444P/L444P hiPSC-macrophages were able to phagocy-
tose opsonized RBC with efficiency similar to that of type 2
GD hiPSC-Macrophage Activation in Response to LPS. To determine
whether GD hiPSC-macrophages would respond to bacterial
products, we treated control and GD hiPSC-macrophages with
the bacterial endotoxin LPS (19). As shown in Fig. 2E, 2-h
treatment of control and type 2 GD hiPSC-macrophages with
LPS induced the production of high levels of TNF-α, IL-10, IL-
12 p35, and IL-12 p40 mRNA. The level of induction of the
inflammatory cytokine TNF-α was significantly higher in GD
hiPSC- than in control hiPSC-macrophages. Future studies
should clarify whether GC deficiency sensitizes GD macro-
phages toward inflammatory responses, as suggested by clinical
observations (3). Taken together, the ability of GD hiPSC-
derived macrophages to phagocyte RBC and to respond to LPS
strongly suggests that they have the functional properties of
GD hiPSC-Macrophages Have Reduced GC Activity and Accumulate
Sphingolipids. As shown in Fig. 3A, the GC enzymatic activity in
N370S/N370S, L444P/L444P, and L444P/RecNciI hiPSC-macro-
phages was less than 5% of that in hiPSC-macrophage controls
from a healthy donor, even though the mutant proteins in the
three types of GD macrophages were still expressed at about 50%
of control levels (Fig. 3A). In patients with GD, cells of the re-
ticuloendothelial system display characteristic lipid accumulation
because of the inability to digest GlcCer derived from normal
metabolism and from the phagocytosis of red and white blood
cells. This accumulation leads to the appearance of lipid-engorged
Gaucher macrophages (20), in which remnants of RBC are often
seen (17, 18, 21, 22). To assess whether GD hiPSC-macrophages
would recapitulate the lipid accumulation seen in macrophages of
patients with GD, we carried out immunofluorescence analysis
using an antibody specific to GlcCer and HPLC-MS/MS. As
shown in Fig. 3 B, a and b, mutant hiPSC-macrophages had ele-
vated levels of GlcCer as compared with control cells (Fig. 3 B, c
and d). HPLC-MS/MS analysis showed that types 2 and 3 GD
hiPSC-macrophages had a 90-fold increase in glucosylsphingosine
as compared with control hiPSC-macrophages, whereas type 1 GD
macrophages exhibited a lower, but still very significant, 28-fold
increase of this lipid (Fig. 3C and Fig. S5). These results show that
(A) Staining of GD hiPSC with antibodies to the stem cell and pluripotency
markers SOX2, SSEA-3, SSEA-4, NANOG, TRA-1–60 TRA-1–81, and OCT4.
(Scale bar, 100 μm.) (B) (a) GD hiPSC cells gave rise to benign cystic teratomas
in NOG-SCID mice. (b–f) H&E staining of teratoma cells from the three germ
layers. (b) Glandular structure. (c) Pigmented neural epithelium and rosettes.
(d) Intestinal epithelium. (e) Cartilage. (f) Bone. (Magnification: 20×.)
Generation and characterization of L444P/RecNciI (type 2) GD hiPSC.
2 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1207889109Panicker et al.
the levels of GC activity in patient-derived hiPSC-macrophages
were not sufficient to catabolize glucosylsphingolipids generated
by normal metabolism in the mutant cells, even in case of the
milder N370S mutant.
Impaired Clearance of RBC in GD hiPSC-Macrophages. When char-
acterizing the phagocytic properties of type 2 GD hiPSC-macro-
phages, we noticed a significant delay in the clearance of ingested
RBC by mutant macrophages compared to control macrophages,
as determined by direct observation under the microscope. The
internalized RBC were visible inside macrophages derived from
both control and GD hiPSC-macrophages for a few hours after
ingestion. However, we noticed that although ingested RBC were
no longer visible in control hiPSC-macrophages after 24 h, rem-
nants of RBC were visible in mutant macrophages even 3 d after
erythrophagocytosis. To confirm these observations, we quantified
the rate of clearance of phagocytosed RBC by GD hiPSC-mac-
rophages. As shown in Fig. 3D, control hiPSC- and hESC-mac-
rophages were able to clear most of the ingested RBC within 1 d.
However, macrophages derived from three different L444P/
RecNciI hiPSC lines exhibited a significant delay in RBC clear-
ance (Fig. 3D). Fig. 3E shows the presence of significant numbers
of engulfed RBC in the L444P/RecNciI type 2 macrophages (Fig.
3 E, a and b) compared with control macrophages (Fig. 3 E, c and
d) 24 h after phagocytosis. L444P/L444P (type 3) and N370S/
N370S (type 1) hiPSC-macrophages also exhibited impaired RBC
clearance, and the extent of the delay in RBC clearance increased
with the severity of the mutation (Fig. 3F). It is worth noting that
the level of sphingolipid accumulation was very similar in type 2
L444P/RecNciI and type 3 L444P/L444P hiPSC-macrophages
(Fig. 3C), suggesting that the RBC clearance assay was better able
to predict clinical subtype than measurement of sphingolipid
Because the presence of RBC remnants is a key feature of
macrophages that infiltrate bone marrow and other organs in
patients with GD (17, 18, 21, 22), the GD hiPSC-macrophages we
obtained recapitulate an important hallmark of the disease. This
abnormal RBC clearance by mutant macrophages might be
caused by blockage of the phagocytic pathway downstream of
RBC uptake, because there were no quantitative differences in the
tograms show the percentage of cells stained with antibodies to specific markers (Lower) and isotype controls (Upper). (A) CD14 expression in GD hiPSC-
monocytes. (B) Expression of CD14 (a), CD163 (b), and CD68 (c) in GD hiPSC-macrophages. (C) May–Grünwald–Giemsa staining showing the appearance of GD
hiPSC-macrophages with different morphologies (a–c). (Scale bar, 20 μm.) (D) (a, b, d, and e) Phagocytosis of opsonized RBC by GD hiPSC-macrophages. (c and f)
Non-opsonized RBC control. (a–c) Live-cell images of GD hiPSC-macrophages. (d–f ), May–Grünwald–Giemsa staining of GD hiPSC-macrophages. (Scale bars,
20 μm.) (E) RT-PCR analysis showing the induction of TNF-α, IL-10, IL-12p35, and IL-12p40 mRNA in response to LPS treatment. Numbers in the ordinates
represent the fold-activation of corresponding cytokines compared with the nontreated condition. P values for fold-cytokine induction in GD hiPSC-macro-
phages compared with control hiPSC-macrophages are P < 0.0138 (TNF-α), P < 0.1 (IL-10), P < 0.0698 (IL-12p35), and P < 0.009 (IL-12p40).
Directed differentiation of type 2 GD hiPSC to monocyte/macrophages. (A and B) FACS analysis of L444P/RecNciI hiPSC-monocyte/macrophages. His-
Panicker et al. PNAS Early Edition
| 3 of 6
rates of RBC ingestion by GD hiPSC-, control hiPSC-, and hESC-
macrophages (Fig. 2D and Fig. S3 E, a and F, a). Future analysis
should clarify the precise steps in the phagocytic cascade that are
blocked, and how this blockage relates to the pathophysiology
Phenotypic Correction of GD hiPSC-Macrophage Defects by Recombinant
GC. As shown in Fig. 3G, treatment of type 2 L444P/RecNciI
hiPSC-macrophages with mannose-exposed recombinant GC re-
stored clearance of phagocytosed RBC in a dose-dependent
manner, to almost the same levels as those in control hiPSC-
macrophages. Recombinant GC also corrected the defect in RBC
clearance in L444P/L444P (type 3) and N370S/N370S (type 1)
mutant macrophages (Fig. S6). GC treatment of control hiPSC-
macrophages caused only a very slight increase in the rate of RBC
clearance. These results suggested that the phenotype we observed
was indeed caused by the GC deficiency and, importantly, that the
uptake of recombinant GC was able to overcome the deleterious
effects of different mutant GC proteins, including the severe
L444P and RecNciI mutations in the type 2 hiPSC-macrophages.
Isofagomine Partially Corrects the Abnormal GD hiPSC-Macrophage
Phenotype. Isofagomine is a competitive inhibitor of GC that
facilitates folding and transport of GC mutants, including the
frequent alleles N370S and L444P (23, 24). Isofagomine has been
evaluated as a possible therapeutic agent for GD in cell lines,
animal models, and clinical trials (24, 25). Isofagomine treatment
of types 1, 2, and 3 GD hiPSC-macrophages increased GC en-
zymatic activity by about 1.7- to 2.0-fold, as compared with 1.3-
fold in control cells (Fig. 3H). To determine whether this drug
would have any effect on RBC clearance by GD hiPSC-
macrophages, we incubated L444P/RecNciI GD hiPSC-macro-
phages with isofagomine for 5 d. After this time, the treated
macrophages were incubated with opsonized RBC, and the rate
of RBC clearance was followed for 3 d in the presence of iso-
fagomine. As shown in Fig. 3I, isofagomine treatment resulted in
a partial increase in the rate of clearance of phagocytosed RBC
by L444P/RecNciI hiPSC-macrophages. Isofagomine treatment
also increased the rates of phagocytosed RBC clearance in
L444P/L444P and N370S/N370 hiPSC-macrophages, but it was
not as effective as recombinant GC in restoring normal RBC
clearance, even in the milder N370S/N370S mutant (Fig. S7D).
Similar results were obtained regardless of whether isofagomine
was added or omitted during the 3-d period following RBC up-
take. Taken together, these results reflect the clinical efficacy of
enzyme replacement therapy (ERT) and isofagomine, and sug-
gest that GD hiPSC and the functional RBC assay described in
this study will be very valuable for evaluating the therapeutic
efficacy of new drugs.
Differentiation of GD hiPSC to the Neuronal Lineage. We then in-
vestigated whether GD hiPSC could be differentiated to neuronal
cell types in vitro. Types 1, 2, and 3 GD hiPSC were differentiated
to neuronal cells efficiently; representative results are shown for
L444P/RecNciI hiPSC in Fig. 4. Neural tube-like rosettes derived
from the neuroepithelial cells expressed markers of neuronal
differentiation, including MAP2 and Tuj1. Fig. 4 A–E shows the
characteristic arrangements of the cells in the rosettes, which
were positive for the neural progenitor marker SOX2 and the
astrocyte marker GFAP (Fig. 4 D and E). In cultures maintained
for over 10–15 d we observed neuronal maturation with estab-
lishment of interneuronal connections, and these cells were
GD-iM + 0.02 U rGCase
GD-iM + 0.04 U rGCase
GD-iM + 0.12 U rGCase
GD-iM + 0.24 U rGCase
GD-iM + 30 µM Isof
GD-iM + 60 µM Isof
GD-iM + 100 µM Isof
Low levels of GC enzymatic activity (Upper) and GC
protein (Lower) in N370S/N370S, L444P/L444P, and
L444P/RecNciI hiPSC- vs. control hiPSC-macrophages
(iMϕ). (B) Staining of L444P/RecNciI (a and b) and
control (c and d) hiPSC-macrophages with rabbit
antibodies to GlcCer (G-Cer, red) and nuclear stain-
ing with DAPI (blue). (C) HPLC-MS/MS analysis
showing the level of glucosylsphingosine in H9/
hESC-, control hiPSC-, N370S/N370S, L444P/L444P, and
phagocytosed RBC clearance in different lines of
L444P/RecNciI mutant macrophages. H9/hESC- (black),
control hiPSC- (red), and three lines of L444P/RecNciI
GD hiPSC-macrophages [#3 (blue), #4 (purple), and
#16 (green)] were incubated with opsonized RBC,
and the time course of RBC clearance was followed.
The ordinate represents the percent of GD hiPSC-
macrophages containing visible RBC. On day 2, P <
0.0001. (E) Significant numbers of phagocytosed RBC
are still visible 24 h after ingestion by L444P/RecNciI
phages (a and b) and control hiPSC-macrophages
(c and d) were incubated with opsonized RBC as
described above, and cells were stained with May–
Grünwald–Giemsa 24 h later. Representative micro-
scopic images are shown. Arrows indicate RBC rem-
nants. (F) Comparison of the time courses of RBC
clearance in type 1 N370S/N370S (blue), type 2 L444P/RecNciI (purple), and type 3 L444P/L444P (green) macrophages, in control hiPSC- (red), and in H9/hESC-
macrophages (black). On day 2, P values corresponding to the type 1, 2, and 3 genotypes compared with the controls were P < 0.008, P < 0.0001, and P < 0.001,
respectively. (G) Phenotypic correction of mutant phenotype by recombinant GC. Untreated control hiPSC-macrophages (purple), untreated L444P/RecNciI hiPSC-
macrophages (blue), and L444P/RecNciI hiPSC-macrophages treated with 0.02 (green), 0.04 (red), 0.08 (light blue), and 0.24 (pink) U/mL recombinant GC, as
described in SI Materials and Methods, were assayed for RBC clearance. On day 2, P values for 0.02, 0.04, 0.08, and 0.24 U/mL GC were P < 0.0015, P < 0.005, P <
0.0005, and P < 0.0001, respectively. (H) Isofagomine treatment increases GC enzyme activity in mutant macrophages. L444P/RecNciI, L444P/L444P, N370S/N370S,
and control hiPSC-macrophages were incubated in the presence or absence of 60 μM isofagomine for 5 d; then GC activity was determined. Numbers represent
fold-increase of GC activity in treated vs. untreated cells. (I) Time course of phagocytosed RBC clearance by GD hiPSC-macrophages in the absence or presence of
isofagomine. L444P/RecNciI hiPSC-macrophages were incubated in the absence (blue) or presence of 30 μM (green), 60 μM (red), or 100 μM (black) isofagomine
for 5 d and then were assayed for RBC clearance as described in D. Untreated H9/hESC macrophages were used as a control (pink). Isofagomine treatment was
continued for the duration of the RBC clearance assay. On day 2, P values for 30 μM and 60 μM isofagomine were P < 0.0002 and P < 0.0006, respectively. (Scale
bars, 20 μm in B and E.)
Phenotype of GD hiPSC-macrophages. (A)
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positive for neuronal-specific markers including Tuj1, MAP2, and
GABA (Fig. 4 F–H). These cultures were also positive for do-
paminergic neuronal markers such as dopamine β hydroxylase
(DβH) and tyrosine hydroxylase (TH) (Fig. 4 I and J). The
rosettes also gave rise to astrocytes and oligodendrocytes, as de-
termined by GFAP and O4 staining, respectively (Fig. 4 E and K).
Importantly, all three types of GD hiPSC-derived neurons had
very low GC enzymatic activity (Fig. 4L) and accumulated glu-
cosylsphingolipids (Fig. S8).
In this study we describe the generation of hiPSC derived from
patients with the three clinical subtypes of GD and their proposed
use for disease modeling and therapeutic evaluation. The cells
used for hiPSC generation were obtained from an adult donor
harboring N370S/N370S (type 1), from a 3-y-old donor with type
3 GD carrying L444P/L444P, and from an infant with type 2 GD
with heteroallelic L444P/RecNciI mutations. L444P is the sec-
ond most frequent mutation among patients with GD, and it is
often associated with the severe type 2 and 3 forms of GD. The
RecNciI allele results from recombination of the GBA gene with
the GBA pseudogene, which harbors an L444P, an A456P, and
a silent V460V mutation (26). In type 2 cases, pathological
abnormalities are already present in utero, resulting in either
fetal mortality or in newborns afflicted by severe visceral and
neurological manifestations (4). We have shown that hiPSC-de-
rived cells from all three types of GD have phenotypic abnor-
malities that increase with the severity of the mutation, and that
in every case recombinant GC can reverse these abnormalities.
The most distinguishing histological finding in GD is the
presence of Gaucher macrophages in the spleen, liver, and bone
marrow, and these cells are believed to be primarily responsible
for the visceral, hematologic, and bone pathology in affected
individuals (1). GD macrophages are engorged with GlcCer, and
GC deficiency also leads to accumulation of glucosylsphingosine,
a sphingolipid that is present in very small quantities in normal
cells but is highly elevated in tissues of patients who have GD
(27). To model the formation of GD macrophages, we carried
out directed differentiation of GD hiPSC in the presence of M-
CSF. The GD hiPSC-macrophages we obtained had elevated
levels of GlcCer and glucosylsphingosine in their resting state.
Thus, the level of mutant GC enzyme was insufficient to catab-
olize glucosylsphingolipids generated by the normal metabolism of
the mutant cells, rendering them particularly susceptible to the
added digestive burden following erythrophagocytosis. One of the
most interesting findings of this study was that GC deficiency
resulted in delayed clearance of phagocytosed RBC by GD hiPSC-
macrophages. Furthermore, we observed an inverse correlation
between the rate of RBC clearance and the severity of the muta-
tion. These observations suggest that the functional assay de-
veloped using hiPSC-macrophages may reflect the extent of
visceral abnormalities in GD patients, and that GD hiPSC can be
very valuable for modeling the molecular and subcellular mecha-
nisms that underlie the pathology of GD. The incomplete digestion
of phagocytosed RBC was not secondary to increased uptake of
RBC by GD hiPSC-macrophages, because we detected no differ-
ence in the rate of erythrophagocytosis between mutant and con-
trol macrophages. Rather, the delay in RBC clearance was
probably caused by blockage of steps that occur after RBC uptake.
Remnants of RBC are frequently observed in Gaucher macro-
phages isolated from patient bone marrow (17, 18, 21, 22), lending
support to the conclusion that the phenotype of GD hiPSC-mac-
rophages that we observed recapitulated that of Gaucher cells.
ERT is the standard of treatment for type 1 GD, and it is also
used to alleviate the visceral and hematologic abnormalities in
types 2 and 3 GD (28). Cerezyme (imiglucerase; Genzyme), the
first enzyme preparation used for ERT, is a recombinant human
GC that is modified by glycosidase treatment to facilitate macro-
phage uptake through mannose receptors. In most patients with
type 1 GD, ERT can reduce the visceral and hematologic in-
volvement within 6 mo of the start of regular enzyme infusions
(29). Our results showed that incubation of hiPSC-macrophages
from all three types of GD with GC restored the clearance of
phagocytosed RBC almost to control levels, even in type 2 hiPSC-
derived macrophages. Thus, administration of normal GC was
able to overcome potential gain-of-function effects of some of the
most harmful GBA mutations. These observations suggest that it
may be possible to identify new therapeutic agents also capable of
reversing the deleterious effects of different GBA mutations. In
future studies with a larger and genotypically diverse GD hiPSC
collection, we will examine whether the in vitro response of in-
duced macrophages to recombinant GC correlates with the clini-
cal efficacy of ERT to alleviate visceral manifestations of GD in
the corresponding donors.
A number of chaperones and other small molecules that have
been tested as potential therapeutic candidates for GD have been
L444P/RecNciI hiPSC were differentiated to neuronal
cells as described in SI Materials and Methods, and
were stained with antibodies to the indicated mark-
ers. (A–E) Characterization of neuronal rosette pro-
genitors. (A) DAPI (blue). (B) MAP2 (red) overlaid
with DAPI. (C) Tuj1 (red). (D) SOX2 (red). (E) GFAP
(green) overlaid with Tuj1 (red). (F–K) Characteriza-
tion of GD hiPSC neuronal cells extending from
rosettes. (F) Tuj1 (green). (G) MAP2 (green). (H)
GABA (green). (I) DβH (green). (J) TH (green). (K) O4
(red) overlaid with DAPI. (L) Low levels of GC enzy-
matic activity in N370S/N370S, L444P/L444P, and
L444P/RecNciI hiPSC- vs. control hiPSC-neurons. (Scale
bars, 100 μm in A–G, 20 μm in H–K.)
Differentiation of GD hiPSC to neuronal cells.
Panicker et al.PNAS Early Edition
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identified in chemical screens using purified GC, followed by
validation using human GD cell lines (30, 31). Isofagomine is
a competitive inhibitor of GC that facilitates folding and transport
of GC mutants, including the frequent N370S and L444P alleles
(32, 33), and has been effective in animal models (24, 25). In
a 6-mo phase II clinical trial, isofagomine administration increased
GC activity in white blood cells in all patients, including homo-
zygous L444P cases, although meaningful clinical improvement
was seen in only 1 of 18 patients (24). Isofagomine treatment of
GD hiPSC-macrophages resulted in increased GC activity, but this
treatment led to only partial restoration of the ability of types 1, 2,
and 3 GD hiPSC-macrophages to clear ingested RBC. Thus, in our
system, isofagomine was less effective than recombinant GC in
correcting the functional defects of GD hiPSC-macrophages har-
boring different GBA mutations, reflecting the clinical experience
with these therapeutic agents. Taken together, our results strongly
suggest that the functional assay reported in this study may be a
very discriminating tool for the assessment of therapeutic efficacy.
However, this conclusion needs to be validated with a larger pa-
tient population for each clinical subtype.
Impaired lysosomal function in GD neurons is likely to in-
terfere with critical cellular functions such as autophagy, in-
tracellular transport, vesicle fusion, and lysosomal clearance of
protein aggregates and organelles targeted for degradation (34,
35). Mutations in GBA have been associated with increased risk
of Parkinson disease (36), and recent studies using primary cells
and GD hiPSC harboring an N370S/84GG genotype (8) showed
that dopaminergic neurons exhibited decreased lysosomal pro-
tein degradation, accumulation of aggregated α-synuclein, and
neurotoxicity (37). In the present study we have shown that GD
hiPSC can be efficiently differentiated to neuronal cell types, and
that the mutant neurons accumulate glucosylsphingolipids. In
future studies, GD hiPSC-neurons will be used for studying the
molecular basis of the neuronopathy found in patients with
In summary, we have described the generation of GD hiPSC,
their directed differentiation to macrophages and neuronal cells,
and have shown their potential to model the functional defects of
GD macrophages. GD hiPSC-macrophages recapitulated the lipid
storage and impaired RBC clearance phenotype of macrophages
infiltrating patient organs (18, 21, 22). In the case of type 2 GD
hiPSC-macrophages, this phenotype was striking. We also showed
that the functional response of the mutant macrophages to ERT
and isofagomine reflected the clinical experience with these ther-
apeutic agents. The generation of GD-hiPSC representative of
types 1, 2, and 3 GD and the ability to differentiate them to the
affected cell types with high efficiency will help elucidate the
mechanisms leading to GD, and will provide a unique platform for
Materials and Methods
Details for the reprogramming of skin fibroblasts from patients with types 1, 2,
and 3 GD, harboring N370S/N370S, L444P/RecNciI, and L444P/L444P mutations
in GC, respectively, into hiPSC using the STEMCCA vector, and their charac-
terization are described in SI Materials and Methods. The methods for dif-
ferentiation of hiPSC to macrophages and neurons and their characterization
by immunofluorescence analysis and flow cytometry also are described in
SI Materials and Methods. The GC enzyme assay, analysis of glucosylsphin-
gosine by HPLC-MS/MS, the erythrophagocytosis assay, and treatments with
recombinant GC and the chaperone isofagomine are described in SI Materials
ACKNOWLEDGMENTS. We thank Gustavo Mostoslavsky (Boston University)
for providing the STEMCCA vector and Avital Shimanovich and Vivek Bose
for expert technical assistance. This work was supported by Grants 2009-
MSCRFII-0082-00 (to R.A.F.), 2007-MSCRFE-0110-00 (to R.A.F.), 2011-MSCRF
II-0008-00 (to E.T.Z.), and 2007-MSCRF II-0379-00 (to E.T.Z.) from the Mary-
land Stem Cell Research Fund (MSCRF), Grant 6-FY10-334 (to R.A.F.) from the
March of Dimes, and Grants 1U01HL099775 and U01HL100397 (both to
E.T.Z.) from the National Institutes of Health. T.S.P. and O.A. are recipients
of MSCRF postdoctoral fellowship grants.
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