論文の公開元へPluripotent stem cell model of Shwachman-Diamond syndrome reveals apoptotic predisposition of hemoangiogenic progenitors
概要
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Pluripotent stem cell model
of Shwachman–Diamond syndrome
reveals apoptotic predisposition
of hemoangiogenic progenitors
Takayuki Hamabata1, Katsutsugu Umeda1*, Kagehiro Kouzuki1, Takayuki Tanaka1,
Tomoo Daifu1, Seishiro Nodomi1, Satoshi Saida1, Itaru Kato 1, Shiro Baba1,
Hidefumi Hiramatsu1, Mitsujiro Osawa2, Akira Niwa2, Megumu K. Saito 2,
Yasuhiko Kamikubo3, Souichi Adachi3, Yoshiko Hashii4, Akira Shimada5,
Hiroyoshi Watanabe6, Kenji Osafune 7, Keisuke Okita 8, Tatsutoshi Nakahata9,
Kenichiro Watanabe10, Junko Takita1 & Toshio Heike1
Shwachman–Diamond syndrome (SDS), an autosomal recessive disorder characterized by bone
marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, is caused by mutations in
the Shwachman–Bodian–Diamond syndrome (SBDS) gene, which plays a role in ribosome biogenesis.
Although the causative genes of congenital disorders frequently involve regulation of embryogenesis,
the role of the SBDS gene in early hematopoiesis remains unclear, primarily due to the lack of a
suitable experimental model for this syndrome. In this study, we established induced pluripotent
stem cells (iPSCs) from patients with SDS (SDS-iPSCs) and analyzed their in vitro hematopoietic and
endothelial differentiation potentials. SDS-iPSCs generated hematopoietic and endothelial cells less
efficiently than iPSCs derived from healthy donors, principally due to the apoptotic predisposition
of KDR+CD34+ common hemoangiogenic progenitors. By contrast, forced expression of SBDS gene
in SDS-iPSCs or treatment with a caspase inhibitor reversed the deficiency in hematopoietic and
endothelial development, and decreased apoptosis of their progenitors, mainly via p53-independent
mechanisms. Patient-derived iPSCs exhibited the hematological abnormalities associated with SDS
even at the earliest hematopoietic stages. These findings will enable us to dissect the pathogenesis of
multiple disorders associated with ribosomal dysfunction.
Shwachman–Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by hematological
abnormalities that manifest as cytopenia and progression to myelodysplastic syndrome and acute myeloid leukemia, exocrine pancreatic insufficiency, and skeletal a bnormalities1,2. Hematopoietic stem cell transplantation
is the sole curative treatment to correct the hematological defect of this syndrome, and mortality remains high
due to serious post-transplant c omplications3.
1
Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara‑cho, Shogoin, Sakyo‑ku,
Kyoto 606‑8507, Japan. 2Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto
University, Kyoto 606‑8507, Japan. 3Department of Human Health Sciences, Graduate School of Medicine, Kyoto
University, Kyoto 606‑8507, Japan. 4Department of Cancer Immunotherapy, Osaka University School of Medicine,
Suita 565‑0871, Japan. 5Department of Pediatric Hematology/Oncology, Okayama University, Okayama 700‑8558,
Japan. 6Department of Pediatrics, Graduate School of Biomedical Sciences, Tokushima University,
Tokushima 770‑8501, Japan. 7Department of Cell Growth and Differentiation, Center for iPS Cell Research and
Application, Kyoto University, Kyoto 606‑8507, Japan. 8Department of Life Science Frontiers, Center for iPS Cell
Research and Application, Kyoto University, Kyoto 606‑8507, Japan. 9Drug Discovery Technology Development
Office, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606‑8507, Japan. 10Department
of Hematology and Oncology, Shizuoka Children’s Hospital, Shizuoka 420‑8660, Japan. *email: umeume@
kuhp.kyoto‑u.ac.jp
Scientific Reports |
(2020) 10:14859
| https://doi.org/10.1038/s41598-020-71844-8
1
Vol.:(0123456789)
www.nature.com/scientificreports/
Mutations in the Shwachman–Bodian–Diamond (SBDS) gene, which is located on chromosome 7q11, are
present in approximately 90% of patients with SDS; these mutations commonly arise from gene conversion to
the highly similar pseudogene SBDSP4. SBDS mRNA is expressed in a broad range of t issues4. Multiple studies
have shown that SBDS protein has a primary function in ribosome a ssembly5–7. The additional proposed functions for SBDS, such as mitotic spindle stabilization, chemotaxis, cellular stress responses, and apoptosis, reflect
indirect downstream effects of perturbing ribosome a ssembly8–13.
Mutations in genes encoding transcription factors involved in regulating normal development are responsible
for a variety of inherited disorders. During embryogenesis, hematopoietic cells (HCs) and endothelial cells (ECs)
emerge from common hemoangiogenic progenitors that express vascular endothelial growth factor receptor
(VEGFR)-2 (also known as KDR in humans)14–16. Indeed, several HC- and/or EC-related transcriptional factors,
such as SCL and RUNX1, are associated with various congenital hematological d
isorders17. Multiple disorders
associated with ribosomal dysfunction (so-called ribosomopathies), including SDS and Diamond–Blackfan
anemia (DBA), also present with hematological defects18; however, the pathogenesis of ribosomopathies has
not been fully elucidated. Currently, the field lacks an adequate mouse model of the human disease because
the most analogous mutant in mouse fails to faithfully recapitulate all disease-associated p
henotype19–22: Sbds-/embryos fail to generate HCs and ECs due to early lethality prior to embryonic day (E) 6.5 before both lineages
have developed23.
Induced pluripotent stem cells (iPSCs) are pluripotent stem cell generated by enforced expression of specific
transcription factors24. Patient-specific iPSCs, in combination with directed cell differentiation, are a practical
source of human embryonic progenitors that can surpass the utility of murine models. Accordingly, these cells
have the potential to contribute enormously to patient-oriented research, including disease pathophysiology
and drug screening25. In this study, we generated iPSCs from three SDS patients and differentiate them into HCs
and ECs using our established differentiation system for human embryonic stem cells (ESCs) and iPSCs26–30.
Results
Generation of iPSCs from SDS patients. Following transduction of peripheral blood cell derived from
SDS patients with an episomal plasmid vector encoding Oct3/4, Sox2, Klf4, L-Myc, Lin28, and shRNA against
TP53, four clones (SDS1-1 and SDS1-2 from patient 1 and SDS2 from patient 2, and SDS3 from patient 3)
were randomly selected for propagation and further analyses, as previously reported29,31. All patient-derived
SDS-iPSCs exhibited a characteristic human ESC-like morphology (Fig. 1a, Supplementary Fig. S1a), and were
capable of propagating in serial passage. DNA sequencing analysis verified an identical mutation in the SBDS
gene in all established SDS-iPSC clones (Fig. 1b, Supplementary Fig. S1b). Chromosomal analysis revealed that
all SDS-iPSC clones maintained a normal karyotype (Fig. 1c, Supplementary Fig. S1c). Expression levels of the
pluripotency markers Oct3/4, Sox2, Klf4, L-Myc, and Lin28 in all SDS-iPSCs were comparable to those in control
iPSCs, although transgene expression was rarely detected (Fig. 1d, Supplementary Fig.S1d). All three primary
germ-layer derivatives were detected in cystic teratomas formed after subcutaneous injection of undifferentiated
iPSCs into immunocompromised NOD/SCID/γcnull mice (Fig. 1e, Supplementary Fig. S1e).
To investigate the pathogenesis of this syndrome, SBDS cDNA and DsRed were transduced into SDS-iPSCs
using the PiggyBac transposon system (Fig. 2a). Western blotting revealed a reduction in SBDS protein expression
in SDS-iPSCs that was rescued in SBDS-overexpressed iPSCs (Fig. 2b). Polysome profiling demonstrated that
ribosomal assembly in SDS-iPSCs was reduced, as evidenced by a decrease in the 80S:40S ratio; this deficiency
was reversed by transduction of SBDS cDNA (Fig. 2c,d).
Impaired granulopoiesis during in vitro differentiation of SDS‑iPSCs. First, using a previously
reported in vitro culture s ystem26,29, we investigated whether generated SDS-iPSCs recapitulated the hematological phenotype of the syndrome (Fig. 3a). Floating HCs, which mainly consisted of mature neutrophils, first
appeared on day 15 of differentiation of SDS and control iPSCs (Fig. 3b,c, Supplementary Fig. S2a). The remaining HCs consisted of immature myeloid cells and a small number of macrophages. Serial analyses revealed that
floating HCs generated from SDS-iPSCs were less abundant than those from control iPSCs (Fig. 3c). Positivity
for myeloperoxidase and lactoferrin, the constituent proteins of neutrophil-specific granules, was comparable in
neutrophils obtained from SDS-iPSCs and control iPSCs (Supplementary Fig. S2b,c). Similarly, the bactericidal
activity of neutrophils from SDS-iPSCs and control iPSCs did not significantly differ (Supplementary Fig. S2d).
HC production was comparable between SBDS-overexpressing SDS-iPSCs and control iPSCs (Fig. 3c,d, Supplementary Fig. S1e,f), with no attendant morphological changes (Supplementary Fig. S2g,h). As reported
previously9,32, the chemotactic activity of SDS-iPSC–derived neutrophils was severely impaired, and this deficiency was reversed by overexpression of SBDS (Fig. 3e).
We then examined the hematological defects of SDS-iPSCs at the clonogenic progenitor level. In methylcellulose colony-forming assays, SDS-iPSCs formed significantly fewer HC colonies, a defect that was rescued by
SBDS overexpression (Fig. 3f). The decreased size of HC colonies was also reversed by SBDS overexpression,
although the size is not somehow comparable to that of control iPSCs (Fig. 3g). Collectively, these data demonstrated that SDS-iPSCs exhibited reduced HC production, accompanied by impaired neutrophil chemotaxis and
limited colony-forming potential, all of which are typical hematological abnormalities of SDS patients. ...
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