Distinct HAND2/HAND2-AS1 Expression Levels May Fine-Tune Mesenchymal and Epithelial Cell Plasticity of Human Mesenchymal Stem Cells

We previously developed several successful decellularization strategies that yielded porcine cardiac extracellular matrices (pcECMs) exhibiting tissue-specific bioactivity and bioinductive capacity when cultured with various pluripotent and multipotent stem cells. Here, we study the tissue-specific effects of the pcECM on seeded human mesenchymal stem cell (hMSC) phenotypes using reverse transcribed quantitative polymerase chain reaction (RT-qPCR) arrays for cardiovascular related gene expression. We further corroborated interesting findings at the protein level (flow cytometry and immunological stains) as well as bioinformatically using several mRNA sequencing and protein databases of normal and pathologic adult and embryonic (organogenesis stage) tissue expression. We discovered that upon the seeding of hMSCs on the pcECM, they displayed a partial mesenchymal-to-epithelial transition (MET) toward endothelial phenotypes (CD31+) and morphologies, which were preceded by an early spike (~Day 3 onward after seeding) in HAND2 expression at both the mRNA and protein levels compared to that in plate controls. The CRISPR-Cas9 knockout (KO) of HAND2 and its associated antisense long non-coding RNA (HAND2-AS1) regulatory region resulted in proliferation arrest, hypertrophy, and senescent-like morphology. Bioinformatic analyses revealed that HAND2 and HAND2-AS1 are highly correlated in expression and are expressed in many different tissue types albeit at distinct yet tightly regulated expression levels. Deviation (downregulation or upregulation) from these basal tissue expression levels is associated with a long list of pathologies. We thus suggest that HAND2 expression levels may possibly fine-tune hMSCs’ plasticity through affecting senescence and mesenchymal-to-epithelial transition states, through yet unknown mechanisms. Targeting this pathway may open up a promising new therapeutic approach for a wide range of diseases, including cancer, degenerative disorders, and aging. Nevertheless, further investigation is required to validate these findings and better understand the molecular players involved, potential inducers and inhibitors of this pathway, and eventually potential therapeutic applications

Disposal of iron by a mutant form of lipocalin 2.

Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe-/-) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway

Disposal of iron by a mutant form of lipocalin 2.

Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe-/-) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway