SummaryOncogenic KRAS mutations in hematopoietic stem cells cause RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). KRAS plays essential roles in stemness maintenance in some types of stem cells. However, its roles in pluripotent stem cells (PSCs) are poorly understood. Here, we investigated the roles of KRAS on stemness in the context of induced PSCs (iPSCs). We used KRAS mutant (G13C/WT) and wild-type isogenic (WT/WT) iPSCs from the same RALD patients, as well as wild-type (WTed/WT) and heterozygous knockout (Δed/WT) iPSCs, both obtained by genome editing from the same G13C/WT clone. Compared with WT iPSCs, G13C/WT iPSCs displayed enforced retention of self-renewal and suppressed capacity for neuronal differentiation, while Δed/WT iPSCs showed normalized cellular characteristics similar to those of isogenic WTed/WT cells. The KRAS-ERK pathway, but not the KRAS-PI3K pathway, was shown to govern these G13C/WT-specific phenotypes, indicating the strong impact of the KRAS-ERK signaling upon self-renewal and differentiation propensity in human iPSCs.
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.
Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.
For the treatment of monogenetic hematological disorders, restoration of transgene expression in affected cell populations is generally considered to have beneficial effects. However, X-linked chronic granulomatous disease (XCGD) is unique since the appearance of functional neutrophils in the peripheral blood following hematopoietic stem cell gene therapy is transient only. One contributing factor could be the occurrence of detrimental effects secondary to ectopic gp91phox expression in neutrophils, which has not been formally demonstrated previously. This study uses iPSCs to model XCGD, which allows the process of differentiation to be studied intensely in vitro. Alpharetroviral vectors carrying a ubiquitous promoter were used to drive the “ectopic” expression of codon optimized gp91phox cDNA. In the mature fraction of neutrophils differentiated from transduced XCGD-iPSCs, cellular recovery in terms of gp91phox expression and reactive oxygen species production was abruptly lost before cells had fully differentiated. Most critically, ectopic gp91phox expression could be identified clearly in the developing fraction of the transduced groups, which appeared to correspond with reduced cell viability. It is possible that this impedes further differentiation of developing neutrophils. Therefore, affording cellular protection from the detrimental effects of ectopic gp91phox expression may improve XCGD clinical outcomes.
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