•Reliable targeting to the AAVS1 requires robust positive selection.•Reporter expression from the CAG promoter is reproducible, scalable, and stable.•Reporter expression is retained following ...differentiation to multiple lineages.•Fluorescent and luminescent iPSCs have value in disease modeling and transplantation studies.
The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.
BackgroundAlthough studies have demonstrated the feasibility of in vivo cardiac transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) using large animals, it requires large ...quantities of purified PSC-CMs. Moreover, genetic modification and contamination of non-CMs are inappropriate for clinical application. Using antibodies on the surface of the transplanted cells is one of the useful methods, but can be immunogenic and cause local inflammation or graft failure. We have shown the synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by microRNAs (miRNAs) expressed in specific cell types can efficiently detect and purify the particular cell populations. Using a miRNA switch and magnetic-activated cell sorting (MACS), we evaluated the efficiency of purification of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in a large scale.MethodsWe used CD4 as a selection marker for MACS and miR-208a as a specific mRNA of CMs. We synthesized CD4 mRNA and transfected it into differentiated cells from iPSCs to confirm CD4 expressing on the surface of the transfected cells. We also synthesized a miRNA switch encoding CD4 tagged with sequences targeted by miR-208a (CD4-208a switch) and transfected it into differentiated cells to demonstrate transfected non-CMs expressing CD4 and transfected CMs non-expressing CD4. Finally, we transfected the CD4-208a switch and puromycin resistance mRNA simultaneously, and purified iPSC-CMs by eliminating CD4+ cells using MACS and untransfected cells using puromycin. Purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium.ResultsAfter transfecting CD4 mRNA into differentiated cells from iPSCs, 78±5% expressed CD4 on the surface. We also confirmed that the CD4-208a switch separated CMs and non-CMs. Using MACS and puromycin selection, we purified iPS-CMs 69±5% to 97±2% assessed by troponin T. Purified cells were also engrafted as CMs in mouse hearts.ConclusionsWe demonstrated that CD4-208a switch purifies iPSC-CMs efficiently in a large scale. Synthetic microRNA switches can apply for many studies of stem cell-based cell replacement therapy for clinical application.
BackgroundAlthough many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments, ...nano-structural confirmation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of engrafted cells in EM has proven to be difficult. However, with a new genetically encoded probe, the monomeric 28-kDa peroxidase reporter 2 (APEX2), which withstands strong EM fixation can resolve this problem. Moreover, immaturity of iPSC-CMs is a critical problem. Especially, excitation-contraction coupling is one of the fundamental properties of cardiomyocytes, the absence of dyad formed between T-tubule and junctional portion of the sarcoplasmic reticulum is one of the major reasons of arrhythmogenic risks after transplantation. Using the APEX2 system, we evaluated morphological alteration of the engrafted iPSC-CMs in EM in a mouse model of myocardial infarction.MethodsWe established human iPSC lines which stably expressed histone H2B-APEX2 (APEX2 iPSCs). After differentiating APEX2 iPSCs into CMs in vitro, purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium. We evaluated the ultrastructure of engrafted iPSC-CMs using EM at 3 and 6 months after transplantation.ResultsAPEX2 did not give significant influences on cardiac differentiation in vitro and stably expressed in iPSC-CMs over 6 months in vivo. APEX2 reaction observed in EM clearly identified engrafted APEX2 iPSC-CMs surrounded by host CMs. The maturation of sarcomeric structure and mitochondria were evident, and T-tubules and dyads started to emerge in engrafted iPSC-CMs at 6 months after transplantation.ConclusionsWe demonstrated that APEX2 is a versatile genetic reporter to trace cell fates in living animals over many months. We unequivocally demonstrated that T-tubules and dyads can be formed in iPSC-CMs after a substantially long period of engraftment. This method should be useful to many studies of stem cell-based cell replacement therapy, as it allows direct nano-scale structural characterization of engrafted cells in EM.
Human induced pluripotent stem cells (iPSCs) are promising cell resources for cell therapy and drug discovery. However, iPSC-derived differentiated cells are often heterogenous and need purification ...using a flow cytometer, which has high cost and time consumption for large-scale purification. MicroRNAs (miRNAs) can be used as cell selection markers, because their activity differs between cell types. Here, we show miRNA-responsive ON and OFF switch mRNAs for robust cell purification. The ON switch contains a miRNA-target sequence after the polyadenylate tail, triggering translational activation by sensing the target miRNA. By designing RNA-only circuits with miRNA-ON and -OFF switch mRNAs that encode a lethal ribonuclease, Barnase, and its inhibitor, Barstar, we efficiently purified specific cell types, including human iPSCs and differentiated cardiomyocytes, without flow cytometry. Synthetic mRNA circuits composed of ON and OFF switches provide a safe, versatile, and time-saving method to purify various cell types for biological and clinical applications.
Human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes are a promising source for regenerative therapy. To realize this therapy, however, their engraftment potential after their injection ...into the host heart should be improved. Here, we established an efficient method to analyze the cell cycle activity of hiPSC cardiomyocytes using a fluorescence ubiquitination-based cell cycle indicator (FUCCI) system. In vitro high-throughput screening using FUCCI identified a retinoic acid receptor (RAR) agonist, Am80, as an effective cell cycle activator in hiPSC cardiomyocytes. The transplantation of hiPSC cardiomyocytes treated with Am80 before the injection significantly enhanced the engraftment in damaged mouse heart for 6 months. Finally, we revealed that the activation of endogenous Wnt pathways through both RARA and RARB underlies the Am80-mediated cell cycle activation. Collectively, this study highlights an efficient method to activate cell cycle in hiPSC cardiomyocytes by Am80 as a means to increase the graft size after cell transplantation into a damaged heart.
For regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to ...collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.
BackgroundAlthough studies have feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments, nano-structural ...confirmation of the successful incorporation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of graft cells in EM has proven to be difficult. However, with a new genetically encoded probe, the monomeric 28-kDa peroxidase reporter 2 (APEX2), which withstands strong EM fixation, this problem can now be done. We have now been able to test whether APEX2 can identify iPSC-CMs in host heart after long-term engrafting, and evaluate the engrafted iPSC-CMs in post-myocardial infarction using EM.MethodsWe established human iPSC lines which stably expressed histone H2B-APEX2 (APEX2 iPSCs). After differentiating APEX2 iPSCs into CM in vitro, purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium. One and 3 months after transplantation, we mapped engraft iPS-CMs using high resolution micro-CT and evaluated their ultrastructure by EM.ResultsAPEX2 was stably expressed and labeled histone H2B in iPSCs before and after in vitro differentiation into CM. Graft efficiency of APEX2 iPSC-CMs in NOG heart was excellent and APEX2 expression sustained over 3 months in vivo. APEX2 reaction observed in EM clearly identified engrafted APEX2 iPSC-CMs in niches surrounded by host CMs and their physical interaction was visualized. EM also revealed a progression in the maturation of sarcomeric structure and mitochondria in engrafted iPSC-CMs, by comparing data at 1 and 3 months after transplantation.ConclusionWe demonstrate that APEX2 is a versatile genetic reporter to trace cell fates in living animals over many months. Using APEX2-based staining, we were able to identify and characterize the maturation process of iPSC-CMs, and determine how they distribute within myocardial niches, as well as their interaction with host CMs. This method should be useful to many studies of stem cell-based cell replacement therapy, as it allows both tracking of cells and the ultrastructural characterization of engrafted cell and graft-host interactions.
Background Patients with type 2 diabetes mellitus (DM) are at high risk for coronary artery disease (CAD), but noninvasive measurement of CAD extent and severity has not been well investigated.
Cardiac regeneration therapy with induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) is a promising therapy to cure drug-resistant heart failure. We have previously reported that ...engrafted iPSC-CMs proliferate for three months after direct injection into the myocardium of immunodeficiency mouse infarcted hearts. We also confirmed the iPSC-CMs is more proliferative in vivo than in vitro. Here, we analyzed the cell cycle of iPSC-CMs to enhance engraftment efficiency after transplantation. We established an iPSC line constitutively expressing Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), which can show the cell cycle phase in real timeCells in a state of S/G2/M phase (proliferating stage) emit green fluorescence, cells in a state of G0/G1 phase (non-proliferating stage) emit red. Next, we compared the engraftment capacity between S/G2/M phase and G0/G1 phase iPSC-CMs, three months after transplantation. Cell cycle activated (S/G2/M phase) iPSC-CMs showed significantly higher engraftment efficiency than those in inactivated (G0/G1 phase). High-throughput screening of compounds for their ability to activate the cell cycle in iPSC-CMs was performed to realize effective engraftment. We extracted some candidates. Second screening identified some group of promising compounds. Among those, we focused on the most effective compound (CCA-1) and optimized the condition of the compound administration. Finally we realized the increase in iPSC-CMs in S/G2/M phase from 2.8% to 39.8% via flow cytometry analysis. We checked the proliferative effect of CCA-1 with other waysEdU & cell number count. EdU assay showed CCA-1 enhanced DNA replication in those iPSC-CMs implying an increased proliferation ability. Similar to the above results, CCA-1 also increased cell number count. We also checked the CCA-1 effect to not FUCCI lines but other cell lines. We confirm EdU assay showed the CCA-1 proliferate CMs in other cell lines in vitro. In vivo, CCA-1 treated iPSC-CMs showed enhanced graft size after transplantation into mice ischemic heart. Altogether, these data demonstrate that cell cycle plays a key role in the effectiveness of cardiac cell therapy using iPSC-CMs, and its modification might lead to novel therapeutic treatments.
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