An assessment of the effects of ectopic gp91phox expression in XCGD iPSC-derived neutrophils

Lin, Huan-Ting; Hara, Masaki; Yamaguchi, Tomoyuki; Wada, Taizo; Yachie, Akihiro; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakauchi, Hiromitsu; Otsu, Makoto

doi:10.1038/mtm.2015.46

Cited by 12 publications

(16 citation statements)

References 43 publications

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“…Some of the hiPSC lines established in this study are already being used for disease studies by our collaborators and our research team, and the results will be reported elsewhere in the near future. For example, we have confirmed that neutrophilic cells differentiated from the TkSCG3 and TkSCG4 lines, derived from X-CGD patients, show severe defects in reactive oxygen species production (data not shown), similar to another X-CGD-iPSC line that we reported previously [39]. Such findings indicate the applicability of the established hiPSC lines to disease-modeling studies.…”

Section: Discussionsupporting

confidence: 87%

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

et al. 2019

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Background Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. Methods We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34 + HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34 + HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34 + -rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4 , OCT4 , SOX2 , and c-MYC . In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. Results We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. Conclusion This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases. Electronic supplementary material The online version of this article (10.1186/s13287-019-1273-2) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 87%

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

et al. 2019

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“…Peripheral blood samples were obtained from a patient with PFE (38-y-old woman) with a nucleotide substitution of 395C>T in the PTH1R gene and a healthy volunteer upon informed consent following the Declaration of Helsinki standard ethics procedure. We generated iPSCs according to previously reported procedures (Lin et al 2015; Kubara et al 2018), with some modifications. Yamanaka factors were introduced into CD34 + hematopoietic stem/progenitor cells isolated from a peripheral blood sample with a Sendai virus SeVdp vector (KOSM302L; Nishimura et al 2017).…”

Section: Methodsmentioning

confidence: 99%

Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption

et al. 2020

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Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients—namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)—using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

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“…However, allogeneic transplants are complicated by graft-versus-host disease and limited donor availability (1). Gene therapy using autologous HSPCs modified by integrating viral vectors has provided clinical benefit but is complicated by vectorrelated genotoxicities and transgene expression driven by exogenous promoters (2)(3)(4). Recent lentiviral vector gene therapy trials have reported clinical benefits without any insertional mutagenesis (5)(6)(7).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease

et al. 2017

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Gene repair of CD34 hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34 HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

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An assessment of the effects of ectopic gp91phox expression in XCGD iPSC-derived neutrophils

Cited by 12 publications

References 43 publications

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption

CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease

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