Telomerase therapy reverses vascular senescence and extends lifespan in progeria mice.

AIMS: Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated ageing syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin) alters nuclear morphology and gene expression. Current therapy increases the lifespan of these children only modestly. Thus, greater understanding of the underlying mechanisms of HGPS is required to improve therapy. Endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) derived from these patients exhibit hallmarks of senescence including replication arrest, increased expression of inflammatory markers, DNA damage, and telomere erosion. We hypothesized that correction of shortened telomeres may reverse these measures of vascular ageing. METHODS AND RESULTS: We generated ECs from iPSCs belonging to children with HGPS and their unaffected parents. Telomerase mRNA (hTERT) was used to treat HGPS ECs. Endothelial morphology and functions were assessed, as well as proteomic and transcriptional profiles with attention to inflammatory markers, DNA damage, and EC identity genes. In a mouse model of HGPS, we assessed the effects of lentiviral transfection of mTERT on measures of senescence, focusing on the EC phenotype in various organs. hTERT treatment of human HGPS ECs improved replicative capacity; restored endothelial functions such as nitric oxide generation, acetylated low-density lipoprotein uptake and angiogenesis; and reduced the elaboration of inflammatory cytokines. In addition, hTERT treatment improved cellular and nuclear morphology, in association with a normalization of the transcriptional profile, effects that may be mediated in part by a reduction in progerin expression and an increase in sirtuin 1 (SIRT1). Progeria mice treated with mTERT lentivirus manifested similar improvements, with a reduction in inflammatory and DNA damage markers and increased SIRT1 in their vasculature and other organs. Furthermore, mTERT therapy increased the lifespan of HGPS mice. CONCLUSION: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases.

Telomerase mRNA Enhances Human Skin Engraftment for Wound Healing.

Deep skin wounds represent a serious condition and frequently require split-thickness skin grafts (STSG) to heal. The application of autologous human-skin-cell-suspension (hSCS) requires less donor skin than STSG without compromising the healing capacity. Impaired function and replicative ability of senescent cutaneous cells in the aging skin affects healing with autologous hSCS. Major determinants of senescence are telomere erosion and DNA damage. Human telomerase reverse transcriptase (hTERT) adds telomeric repeats to the DNA and can protect against DNA damage. Herein, hTERT mRNA lipid nanoparticles (LNP) are proposed and evaluated for enhancing cellular engraftment and proliferation of hSCS. Transfection with optimized hTERT mRNA LNP system enables delivery and expression of mRNA in vitro in keratinocytes, fibroblasts, and in hSCS prepared from donors' skin. Telomerase activity in hSCS is significantly increased. hTERT mRNA LNP enhance the generation of a partial-thickness human skin equivalent in the mouse model, increasing hSCS engraftment (Lamin) and proliferation (Ki67), while reducing cellular senescence (p21) and DNA damage (53BP1).

MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells.

Ethanol exposure during pregnancy is an established cause of birth defects, including neurodevelopmental defects. Most adult neurons are produced during the second trimester-equivalent period. The fetal neural stem cells (NSCs) that generate these neurons are an important but poorly understood target for teratogenesis. A cohort of miRNAs, including miR-153, may serve as mediators of teratogenesis. We previously showed that ethanol decreased, while nicotine increased miR-153 expression in NSCs. To understand the role of miR-153 in the etiology of teratology, we first screened fetal cortical NSCs cultured ex vivo, by microarray and quantitative RT-PCR analyses, to identify cell-signaling mRNAs and gene networks as important miR-153 targets. Moreover, miR-153 over-expression prevented neuronal differentiation without altering neuroepithelial cell survival or proliferation. Analysis of 3'UTRs and in utero over-expression of pre-miR-153 in fetal mouse brain identified Nfia (nuclear factor-1A) and its paralog, Nfib, as direct targets of miR-153. In utero ethanol exposure resulted in a predicted expansion of Nfia and Nfib expression in the fetal telencephalon. In turn, miR-153 over-expression prevented, and partly reversed, the effects of ethanol exposure on miR-153 target transcripts. Varenicline, a partial nicotinic acetylcholine receptor agonist that, like nicotine, induces miR-153 expression, also prevented and reversed the effects of ethanol exposure. These data collectively provide evidence for a role for miR-153 in preventing premature NSC differentiation. Moreover, they provide the first evidence in a preclinical model that direct or pharmacological manipulation of miRNAs have the potential to prevent or even reverse effects of a teratogen like ethanol on fetal development.

CD24 expression identifies teratogen-sensitive fetal neural stem cell subpopulations: evidence from developmental ethanol exposure and orthotopic cell transfer models.

BACKGROUND: Ethanol is a potent teratogen. Its adverse neural effects are partly mediated by disrupting fetal neurogenesis. The teratogenic process is poorly understood, and vulnerable neurogenic stages have not been identified. Identifying these is a prerequisite for therapeutic interventions to mitigate effects of teratogen exposures. METHODS: We used flow cytometry and qRT-PCR to screen fetal mouse-derived neurosphere cultures for ethanol-sensitive neural stem cell (NSC) subpopulations, to study NSC renewal and differentiation. The identity of vulnerable NSC populations was validated in vivo, using a maternal ethanol exposure model. Finally, the effect of ethanol exposure on the ability of vulnerable NSC subpopulations to integrate into the fetal neurogenic environment was assessed following ultrasound guided, adoptive transfer. RESULTS: Ethanol decreased NSC mRNAs for c-kit, Musashi-1and GFAP. The CD24(+) NSC population, specifically the CD24(+)CD15(+) double-positive subpopulation, was selectively decreased by ethanol. Maternal ethanol exposure also resulted in decreased fetal forebrain CD24 expression. Ethanol pre-exposed CD24(+) cells exhibited increased proliferation, and deficits in cell-autonomous and cue-directed neuronal differentiation, and following orthotopic transplantation into naïve fetuses, were unable to integrate into neurogenic niches. CD24(depleted) cells retained neurosphere regeneration capacity, but following ethanol exposure, generated increased numbers of CD24(+) cells relative to controls. CONCLUSIONS: Neuronal lineage committed CD24(+) cells exhibit specific vulnerability, and ethanol exposure persistently impairs this population's cell-autonomous differentiation capacity. CD24(+) cells may additionally serve as quorum sensors within neurogenic niches; their loss, leading to compensatory NSC activation, perhaps depleting renewal capacity. These data collectively advance a mechanistic hypothesis for teratogenesis leading to microencephaly.