Human Genomic Safe Harbors and the Suicide Gene-Based Safeguard System for iPSC-Based Cell Therapy.

The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell-based therapy. However, there are concerns that must be addressed prior to their broad clinical applications and a major concern is tumorigenicity. Suicide gene approaches could eliminate wayward tumor-initiating cells even after cell transplantation, but their efficacy remains controversial. Another concern is the safety of genome editing. Our knowledge of human genomic safe harbors (GSHs) is still insufficient, making it difficult to predict the influence of gene integration on nearby genes. Here, we showed the topological architecture of human GSH candidates, AAVS1, CCR5, human ROSA26, and an extragenic GSH locus on chromosome 1 (Chr1-eGSH). Chr1-eGSH permitted robust transgene expression, but a 2 Mb-distant gene within the same topologically associated domain showed aberrant expression. Although knockin iPSCs carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were sufficiently sensitive to ganciclovir in vitro, the resulting teratomas showed varying degrees of resistance to the drug in vivo. Our findings suggest that the Chr1-eGSH is not suitable for therapeutic gene integration and highlight that topological analysis could facilitate exploration of human GSHs for regenerative medicine applications. Our data indicate that the HSV-TK/ganciclovir suicide gene approach alone may be not an adequate safeguard against the risk of teratoma, and suggest that the combination of several distinct approaches could reduce the risks associated with cell therapy. Stem Cells Translational Medicine 2019;8:627&638.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Thrombopoietin-induced CXC chemokines, NAP-2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation.

BACKGROUND: We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide-2 (NAP-2) and platelet factor-4 (PF-4), were induced by megakaryocyte-specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved. RESULTS: NAP-2 and PF-4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c-Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP-2 and PF-4 inhibited polyploidization of c-Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c-Kit+CD41+ cells. CONCLUSIONS: NAP-2 and PF-4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.

Isolation and characterization of mouse homolog of the neutrophil activating peptide-2.

In the presence of thrombopoietin (TPO), megakaryocytes mature by polyploidization and cytoplasmic maturation, and the matured megakaryocytes induce drastic morphological change and proplatelet formation and release a number of platelets. However, the regulatory mechanism of this unique differentiation process is still obscure. We therefore attempted to identify the factors, expression of which is induced by TPO stimulation in mouse bone marrow megakaryocytes. We isolated the mouse homolog of the neutrophil activating peptide-2 (NAP-2). Mouse NAP-2 cDNA encodes a predicted sequence of 113 amino acids and contains the Cys motif (CXC) found in other members of the alpha-chemokine family. At the amino acid level, the predicted mouse NAP-2 has 50.4%, 51.8%, and 72.6% identity with the predicted human, pig, and rat NAP-2, respectively. Northern blot analysis demonstrates that mouse NAP-2 is expressed only in spleen. Furthermore, the RT-PCR technique shows that the mouse NAP-2 gene is clearly upregulated by TPO stimulation in mouse megakaryocytes.