c-Myc/microRNA-17-92 Axis Phase-Dependently Regulates PTEN and p21 Expression via ceRNA during Reprogramming to Mouse Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are promising cell sources for regenerative medicine and disease modeling. iPSCs are commonly established by introducing the defined reprogramming factors Oct4, Sox2, Klf4, and c-Myc. However, iPSC reprogramming efficiency remains low. Although recent studies have identified microRNAs that contribute to efficient reprogramming, the underlying molecular mechanisms are not completely understood. miR-17-92 is highly expressed in embryonic stem cells and may play an important role in regulating stem cell properties. Therefore, we examined the role of miR-17-92 in the induction of mouse iPSC production. c-Myc-mediated miR-17-92 upregulation increased reprogramming efficiency, whereas CRISPR/Cas9-based deletion of the miR-17-92 cluster decreased reprogramming efficiency. A combination of in silico and microarray analyses revealed that Pten and cyclin-dependent kinase inhibitor 1 (known as p21) are common target genes of miR-17 and miR-20a, which are transcribed from the miR-17-92 cluster. Moreover, miR-17-92 downregulated p21 in the early phase and PTEN in the mid-to-late phase of reprogramming. These downregulations were perturbed by introducing the 3' UTR of PTEN and p21, respectively, suggesting that PTEN and p21 mRNAs are competing endogenous RNAs (ceRNA) against miR-17-92. Collectively, we propose that the c-Myc-mediated expression of miR-17-92 is involved in iPSC reprogramming through the phase-dependent inhibition of PTEN and p21 in a ceRNA manner, thus elucidating an underlying mechanism of iPSC reprogramming.

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.

Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding.

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.

Metabolic remodeling during somatic cell reprogramming to induced pluripotent stem cells: involvement of hypoxia-inducible factor 1.

Induced pluripotent stem cells (iPSCs) were first established from differentiated somatic cells by gene introduction of key transcription factors, OCT4, SOX2, KLF4, and c-MYC, over a decade ago. Although iPSCs can be applicable for regenerative medicine, disease modeling and drug screening, several issues associated with the utilization of iPSCs such as low reprogramming efficiency and the risk of tumorigenesis, still need to be resolved. In addition, the molecular mechanisms involved in the somatic cell reprogramming to pluripotency are yet to be elucidated. Compared with their somatic counterparts, pluripotent stem cells, including embryonic stem cells and iPSCs, exhibit a high rate of glycolysis akin to aerobic glycolysis in cancer cells. This is known as the Warburg effect and is essential for maintaining stem cell properties. This unique glycolytic metabolism in iPSCs can provide energy and drive the pentose phosphate pathway, which is the preferred pathway for rapid cell proliferation. During reprogramming, somatic cells undergo a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis trigged by a transient OXPHOS burst, resulting in the initiation and progression of reprogramming to iPSCs. Metabolic intermediates and mitochondrial functions are also involved in the epigenetic modification necessary for the process of iPSC reprogramming. Among the key regulatory molecules that have been reported to be involved in metabolic shift so far, hypoxia-inducible factor 1 (HIF1) controls the transcription of many target genes to initiate metabolic changes in the early stage and maintains glycolytic metabolism in the later phase of reprogramming. This review summarizes the current understanding of the unique metabolism of pluripotent stem cells and the metabolic shift during reprogramming, and details the relevance of HIF1 in the metabolic shift.

STAT3 for Cardiac Regenerative Medicine: Involvement in Stem Cell Biology, Pathophysiology, and Bioengineering.

Heart disease is the most common cause of death in developed countries, but the medical treatments for heart failure remain limited. In this context, the development of cardiac regeneration therapy for severe heart failure is important. Owing to their unique characteristics, including multiple differentiation and infinitive self-renewal, pluripotent stem cells can be considered as a novel source for regenerative medicine. Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling plays critical roles in the induction, maintenance, and differentiation of pluripotent stem cells. In the heart, JAK/STAT3 signaling has diverse cellular functions, including myocardial differentiation, cell cycle re-entry of matured myocyte after injury, and anti-apoptosis in pathological conditions. Therefore, regulating STAT3 activity has great potential as a strategy of cardiac regeneration therapy. In this review, we summarize the current understanding of STAT3, focusing on stem cell biology and pathophysiology, as they contribute to cardiac regeneration therapy. We also introduce a recently reported therapeutic strategy for myocardial regeneration that uses engineered artificial receptors that trigger endogenous STAT3 signal activation.

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.