Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.

BACKGROUND: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling. METHODS: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment. RESULTS: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/ß-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8. CONCLUSION: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera.

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling.

Chimeric G-CSF Receptor-Mediated STAT3 Activation Contributes to Efficient Induction of Cardiomyocytes from Mouse Induced Pluripotent Stem Cells.

Producing a sufficient number of cardiomyocytes from pluripotent stem cells has been of great demand for cardiac regeneration therapy. However, it remains challenging to efficiently differentiate cardiomyocytes with low costs. Reportedly, granulocyte colony-stimulating factor (G-CSF) receptor (GCSFR) signaling activates signal transducers and activators of transcription (STAT) signaling and enhances cardiac differentiation from embryonic stem cells or induced pluripotent stem cells (iPSCs). To economically and efficiently produce cardiomyocytes from iPSCs through GCSFR/STAT axis activation, we constructed antibody/receptor chimeras that can respond to an inexpensive small molecule. Single-chain Fv of anti-fluorescein (FL) antibody was ligated to transmembrane/cytoplasmic domains of GCSFRs, enabling transduction of GCSFR signaling in response to FL-conjugated bovine serum albumin (BSA-FL) as an alternative ligand. Mouse iPSC lines constitutively expressing these chimeric receptors exhibited increased BSA-FL-induced STAT3 phosphorylation in a dose-dependent manner, which was abolished by an inhibitor of Janus tyrosine kinase (JAK). In addition, BSA-FL stimulation also increased the incidence of beating embryoid bodies and upregulated cardiac-specific gene expressions after differentiation in these iPSC lines. Therefore, the chimeric GCSFRs activated endogenous GCSFR signaling at least via the JAK/STAT3 pathway, thereby enhancing cardiac differentiation from iPSCs. This approach, as an economical strategy, could contribute to stem cell-based cardiac regeneration therapy.

T cell growth control using hapten-specific antibody/interleukin-2 receptor chimera.

IL-2 is a cytokine that is essential for the expansion and survival of activated T cells. Although adoptive transfer of tumor-specific T cells with IL-2 is one of strategies for cancer immunotherapy, it is essential to replace IL-2 that exerts severe side effects in vivo. To solve this problem, we propose to use an antibody/IL-2R chimera, which can transduce a growth signal in response to a cognate antigen. We constructed two chimeras, in which ScFv of anti-fluorescein antibody was tethered to extracellular D2 domain of erythropoietin receptor and transmembrane/cytoplasmic domains of IL-2Rbeta or gamma chain. When the chimeras were co-expressed in IL-3-dependent pro-B cell line Ba/F3 and IL-2-dependent T cell line CTLL-2, gene-modified cells were selectively expanded in the absence of IL-3 and IL-2, respectively, by adding fluorescein-conjugated BSA (BSA-FL) as a cognate antigen. Growth assay revealed that the cells with the chimeras transduced a growth signal in a BSA-FL dose-dependent manner. Furthermore, STAT3, STAT5, ERK1/2 and Akt, which are hallmarks for IL-2R signaling, were all activated by the chimeras in CTLL-2 transfectant. We also demonstrated that the chimeras were functional in murine primary T cells. These results demonstrate that the antibody/IL-2R chimeras could substantially mimic the wild-type IL-2R and could specifically expand gene-modified T cells in the presence of the cognate antigen.

Antigen-mediated growth control of hybridoma cells via a human artificial chromosome.

Human artificial chromosome (HAC) vectors possess several characteristics sufficient for the requirements of gene therapy vectors, including stable episomal maintenance and mediation of long-term transgene expression. In this study, we adopted an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/cytokine receptor chimera that triggers a growth signal in response to a cognate non-toxic antigen, and applied it to growth control of HAC-transferred cells by adding an antigen that differed from cytokines that may manifest pleiotropic effects. We previously constructed a novel HAC vector, 21 Delta qHAC, derived from human chromosome 21, housed in CHO cells. Here, we constructed an HAC vector harboring an ScFv-gp130 chimera responsive to fluorescein-conjugated BSA (BSA-FL) as well as a model transgene, enhanced green fluorescent protein (EGFP), in CHO cells. The modified HAC was transferred into interleukin (IL)-6-dependent hybridoma 7TD1 cells by microcell-mediated chromosome transfer, and the cells were subsequently found to show BSA-FL-dependent cell growth and sustained expression of EGFP in the absence of IL-6. The AMEGA system in combination with HAC technology will be useful for increasing the efficacy of gene therapy by conferring a growth advantage on the genetically modified cells.

Selective expansion of genetically modified T cells using an antibody/interleukin-2 receptor chimera.

Although adoptive transfer of tumor-specific T cells is a plausible approach for cancer immunotherapy, the therapeutic application was hampered due to severe side effects caused by administration of high-dose interleukin (IL)-2, which was used for long-lasting maintenance of tumor-specific T cells in vivo. To solve this problem, here we propose to use an antibody/IL-2 receptor chimera, which can transduce a growth signal in response to a cognate antigen. As a model system, V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was tethered to extracellular D2 domain of erythropoietin receptor and transmembrane/cytoplasmic domains of IL-2 receptor beta or gamma chain. When the pairs of chimeric receptors (V(H)-IL-2Rbeta and V(L)-IL-2Rgamma, or V(H)-IL-2Rgamma and V(L)-IL-2Rbeta) were expressed in IL-3-dependent pro-B cell line Ba/F3 and IL-2-dependent T cell line CTLL-2, the cognate antigen HEL induced selective expansion of gene-modified cells in the absence of IL-3 and IL-2, respectively. Growth assay revealed that the combination of V(H)-IL-2Rbeta and V(L)-IL-2Rgamma transduced a more stringent HEL-dependent growth signal, indicating some conformational effects of the chimeras. Furthermore, STAT3, STAT5 and ERK1/2, which are hallmarks for IL-2R signaling, were all activated by the antibody/IL-2R chimeras. These results clearly demonstrate that the antibody/IL-2R chimeras could substantially mimic the wild-type IL-2R signaling, suggesting the potential application in expansion of gene-modified T cells.

Antigen-mediated migration of murine pro-B Ba/F3 cells via an antibody/receptor chimera.

Cell migration is one of the fundamental cellular responses governing development, homeostasis and disorders of the body. Therefore, artificial control of cell migration holds great promise for the treatment of many diseases. In this study, we developed an artificial cell migration system based on chimeric receptors that can respond to an artificial ligand that is quite different from natural chemoattractants. Chimeric receptors consisting of an anti-fluorescein single-chain Fv tethered to the extracellular D2 domain of erythropoietin receptor (EpoR) and the transmembrane/cytoplasmic domains of EpoR, gp130, interleukin-2 receptor, c-Kit, c-Fms, epidermal growth factor receptor (EGFR) or insulin receptor were expressed in the murine Ba/F3 pro-B cell line. Migration assays revealed that chimeric receptors containing the cytoplasmic domain of c-Kit, c-Fms or EGFR transduced migration signals in response to fluorescein-conjugated bovine serum albumin (BSA-FL). Furthermore, based on the cell migration in response to BSA-FL, we successfully selected genetically modified cells from mixtures of gene-transduced and untransduced cells. This study represents the first demonstration of cell migration in response to an artificial ligand that is quite different from natural chemoattractants, suggesting its potential application to immunotherapies and tissue engineering.

ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency.

Cell metabolism is adaptive to extrinsic demands; however, the intrinsic metabolic demands that drive the induced pluripotent stem cell (iPSC) program remain unclear. Although glycolysis increases throughout the reprogramming process, we show that the estrogen-related nuclear receptors (ERRα and ERRγ) and their partnered co-factors PGC-1α and PGC-1ß are transiently induced at an early stage, resulting in a burst of oxidative phosphorylation (OXPHOS) activity. Upregulation of ERRα or ERRγ is required for the OXPHOS burst in both human and mouse cells, respectively, as well as iPSC generation itself. Failure to induce this metabolic switch collapses the reprogramming process. Furthermore, we identify a rare pool of Sca1(-)/CD34(-) sortable cells that is highly enriched in bona fide reprogramming progenitors. Transcriptional profiling confirmed that these progenitors are ERRγ and PGC-1ß positive and have undergone extensive metabolic reprogramming. These studies characterize a previously unrecognized, ERR-dependent metabolic gate prior to establishment of induced pluripotency.

Foxd1 is a mediator and indicator of the cell reprogramming process.

It remains unclear how changes in gene expression profiles that establish a pluripotent state are induced during cell reprogramming. Here we identify two forkhead box transcription factors, Foxd1 and Foxo1, as mediators of gene expression programme changes during reprogramming. Knockdown of Foxd1 or Foxo1 reduces the number of iPSCs, and the double knockdown further reduces it. Knockout of Foxd1 inhibits downstream transcriptional events, including the expression of Dax1, a component of the autoregulatory network for maintaining pluripotency. Interestingly, the expression level of Foxd1 is transiently increased in a small population of cells in the middle stage of reprogramming. The transient Foxd1 upregulation in this stage is correlated with a future cell fate as iPSCs. Fate mapping analyses further reveal that >95% of iPSC colonies are derived from the Foxd1-positive cells. Thus, Foxd1 is a mediator and indicator of successful progression of reprogramming.