Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell ...populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >10
-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only ...small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
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•AAV6 is an effective donor delivery vector for genome editing in hPSCs•Electroporation of Cas9 RNP prior to AAV6 transduction yields editing up to 90%•The Cas9 RNP/AAV6 method allows for specific modifications ranging from 1 to >3,000 bp•This method yields highly edited cells without selection markers or antibiotics
Matthew Porteus and colleagues report a method for high-frequency genome editing in human pluripotent stem cells, enabling introduction of site-specific modifications ranging from single-base-pair changes to 3-kb integrations without the use of fluorescent markers or antibiotic selection.
Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the ...injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.
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•EpiSCs injected into blastocysts rapidly disappear due to apoptosis•Induced BCL2 expression enables injected EpiSCs to survive and form chimeras•BCL2-expressing Sox17+ endoderm progenitors can also form region-specific chimeras•BCL2-expressing rat EpiSCs form interspecies chimeras that survive to adulthood
Masaki et al. show that expression of the anti-apoptotic gene BCL2 allows developmentally incompatible cells such as epiblast stem cells and endoderm progenitors to engraft into mouse blastocysts. This approach can overcome stage-related barriers to cellular integration, allowing effective formation of chimeras within and between species.
Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within ...3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.
The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a ...major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.
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•iPSC-derived rejuvenated CTLs are effective against EBV-induced tumors in vivo•Rejuvenated CTLs are implemented with an inducible caspase-9 (iC9)-based suicide system•Upon induction, the iC9 system efficiently leads to apoptosis in rejuvenated CTLs•The iC9-based system provides a safeguard for future iPSC-mediated cell therapy
In this article, Nakauchi and colleagues show that iPSC-derived rejuvenated CTLs (rejCTLs) implemented with an inducible caspase-9 (iC9)-based suicide system are effective against EBV-induced tumors in vivo. Upon induction, the iC9-based system efficiently and safely leads to apoptosis in these rejCTLs in vitro and in vivo and provides a safeguard for future iPSC-mediated cell therapy.
Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple ...leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs. Stepwise assembly of the gene segments seems to occur by replacement of the intervening DNA between the 5' and 3' constant-region genes. Here we report that lamprey (Lethenteron japonicum) assemble their VLR genes by a process involving 'copy choice'. Regions of short homology seemed to prime copying of donor LRR-encoding sequences into the recipient gene. Those LRR-encoding germline sequences were abundant and shared extensive sequence homologies. Such genomic organization permits initiation of copying anywhere in an LRR-encoding module for the generation of various hybrid LRRs. Thus, a vast repertoire of recombinant VLR genes could be generated not only by copying of various LRR segments in diverse combinations but also by the use of multiple sites in an LRR gene segment for priming.
Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable ...maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.
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•Different BSA lots alter how HSCs respond to cytokines•RSA can replace BSA to provide HSC maintenance culture with minimal variability•By comparing the protein profiles of “good” and “bad” BSAs, HPX was identified•HPX reduces HSC intracellular reactive ROS and is expressed by BM Schwann cells
In this article, Yamazaki, Nakauchi, and colleagues demonstrate that BSA batches have unique protein profiles and vary widely in their ability to maintain mouse HSCs ex vivo. By replacing BSA with recombinant serum albumin, they developed a standardized HSC culture platform that can be used to identify novel maintenance and expansion factors.
The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted ...to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+)CD41(+)CD45(-) phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.
Although it is important to clarify the pathogenic functions of T cells in human samples, their examination is often limited due to difficulty in obtaining sufficient numbers of dendritic cells ...(DCs), used as antigen-presenting cells, especially in autoimmune diseases. We describe the generation of DCs from induced pluripotent stem cells derived from T cells (T-iPSCs). We reprogrammed CD4+ T cell clones from a patient with Sjögren's syndrome (SS) into iPSCs, which were differentiated into DCs (T-iPS-DCs). T-iPS-DCs had dendritic cell-like morphology, and expressed CD11c, HLA-DR, CD80, CD86, and also BDCA-3. Compared with monocyte-derived DCs, the capacity for antigen processing was similar, and T-iPS-DCs induced the proliferative response of autoreactive CD4+ T cells. Moreover, we could evaluate T cell functions of the patient with SS. In conclusion, we obtained adequate numbers of DCs from T-iPSCs, which could be used to characterize pathogenic T cells in autoimmune diseases such as SS.
•Dendritic cells were generated from iPSCs derived from CD4+ T cells (T-iPS-DCs)•Adequate numbers of functional DCs were generated from a small blood sample•The comparison between T-iPS-DCs and monocyte-derived DCs was evaluated•The functional assays of T cells in Sjögren's syndrome were analyzed by T-iPS-DCs
In this article, Sumida and colleagues demonstrate the generation of iPSCs from CD4+ T cell clones of Sjögren's syndrome (SS), which differentiated into dendritic cells (DCs) (T-iPS-DCs). Adequate numbers of functional DCs were obtained and used for the analysis of T cell functions in SS. T-iPS-DCs could be used to characterize pathogenic T cells in autoimmune diseases such as SS.
Thirty-nine patients with endometrioid adenocarcinoma (EA) and atypical hyperplasia (AH) of the endometrium who received conservative treatment to preserve fertility were collected from member ...institutions of the Japan Gynecologic Oncology Study Group. Twenty-nine and ten were originally diagnosed with EA without myometrial invasion and AH, respectively. We performed a central pathological review to make definite diagnoses, and the diagnosis of EA in 29 cases was changed to AH in ten, complex hyperplasia in three and atypical polypoid adenomyoma in three, and AH in ten was changed to EA in one and simple hyperplasia in one. Nine of 12 women (75%) with EA and 15 of 18 women (83%) with AH had an initial response to medroxyprogesterone acetate (MPA) treatment. Two of nine responders with EA later developed relapse, and one of them had metastasis to the left obturator lymph node. Two became pregnant, and one delivered one full-term infant. One of the responders with AH had a relapse in the endometrium. Five became pregnant, and four delivered four normal infants. The young women with endometrial carcinoma localized in the endometrium who wish to preserve fertility may be treated as successfully with MPA as those with AH.
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