NANOG Reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF-Dependent Gene Expression.

Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)-ß pathways-both of which were impaired in senescent cells-leading to ACTIN polymerization, MRTF-A translocation into the nucleus and serum response factor (SRF)-dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. Stem Cells 2017;35:207-221.

The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling.

Protein arginine methylation regulates diverse functions of eukaryotic cells, including gene expression, the DNA damage response, and circadian rhythms. We showed that arginine residues within the third intracellular loop of the human D2 dopamine receptor, which are conserved in the DOP-3 receptor in the nematode Caenorhabditis elegans, were methylated by protein arginine methyltransferase 5 (PRMT5). By mutating these arginine residues, we further showed that their methylation enhanced the D2 receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) signaling in cultured human embryonic kidney (HEK) 293T cells. Analysis of prmt-5-deficient worms indicated that methylation promoted the dopamine-mediated modulation of chemosensory and locomotory behaviors in C. elegans through the DOP-3 receptor. In addition to delineating a previously uncharacterized means of regulating GPCR (heterotrimeric guanine nucleotide-binding protein-coupled receptor) signaling, these findings may lead to the development of a new class of pharmacological therapies that modulate GPCR signaling by changing the methylation status of these key proteins.

Magnetofection Mediated Transient NANOG Overexpression Enhances Proliferation and Myogenic Differentiation of Human Hair Follicle Derived Mesenchymal Stem Cells.

We used magnetofection (MF) to achieve high transfection efficiency into human mesenchymal stem cells (MSCs). A custom-made magnet array, matching well-to-well to a 24-well plate, was generated and characterized. Theoretical predictions of magnetic force distribution within each well demonstrated that there was no magnetic field interference among magnets in adjacent wells. An optimized protocol for efficient gene delivery to human hair follicle derived MSCs (hHF-MSCs) was established using an egfp-encoding plasmid, reaching approximately ∼50% transfection efficiency without significant cytotoxicity. Then we applied the optimized MF protocol to express the pluripotency-associated transcription factor NANOG, which was previously shown to reverse the effects of organismal aging on MSC proliferation and myogenic differentiation capacity. Indeed, MF-mediated NANOG delivery increased proliferation and enhanced the differentiation of hHF-MSCs into smooth muscle cells (SMCs). Collectively, our results show that MF can achieve high levels of gene delivery to MSCs and, therefore, may be employed to moderate or reverse the effects of cellular senescence or reprogram cells to the pluripotent state without permanent genetic modification.

Differential effects of culture senescence and mechanical stimulation on the proliferation and leiomyogenic differentiation of MSC from different sources: implications for engineering vascular grafts.

We examined the effects of senescence on the proliferation and leiomyogenic differentiation potential of mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs) or hair follicles (HF-MSCs). To this end, we compared ovine HF-MSCs and BM-MSCs in terms of their proliferation and differentiation potential to the smooth muscle cell lineage. We discovered that HF-MSCs are less susceptible to culture senescence compared with BM-MSCs. We hypothesized that application of mechanical forces may enhance the contractility and mechanical properties of vascular constructs prepared from senescent MSCs. Interestingly, HF-MSCs and BM-MSCs responded differently to changes in the mechanical microenvironment, suggesting that despite phenotypic similarities, MSCs from different anatomic locations may activate different pathways in response to the same microenvironmental factors. In turn, this may also suggest that cell-based tissue regeneration approaches may need to be tailored to the stem cell origin, donor age, and culture time for optimal results.

Lentiviral arrays for live-cell dynamic monitoring of gene and pathway activity during stem cell differentiation.

Uncovering the complexity of mesenchymal stem cell (MSC) differentiation requires novel methods to capture the dynamics of the process in a quantitative and high-throughput manner. To this end, we developed a lentiviral array (LVA) of reporters to capture the dynamics of gene and pathway activity during MSC differentiation into adipogenic, chondrogenic, and osteogenic lineages. Our results identified signature promoters and pathways with unique activation profile for each MSC lineage. In combination with chemical inhibitors, lineage-specific reporters predicted the effects of signaling pathway perturbations on MSC differentiation. Interestingly, some pathways were critical for differentiation into all lineages, while others had differential effects on each lineage. Our study suggests that when combined with large chemical or siRNA libraries, the reporter LVA can be used to uncover novel genes and signaling pathways affecting complex biological processes such as stem cell differentiation or reprogramming.

Differential and synergistic effects of mechanical stimulation and growth factor presentation on vascular wall function.

We investigated the hypothesis that immobilizing TGF-ß1 within fibrin hydrogels may act in synergy with cyclic mechanical stimulation to enhance the properties of vascular grafts. To this end, we engineered a fusion TGF-ß1 protein that can covalently anchor to fibrin during polymerization upon the action of factor XIII. We also developed a 24-well based bioreactor in which vascular constructs can be mechanically stimulated by distending the silastic mandrel in the middle of each well. TGF-ß1 was either conjugated to fibrin or supplied in the culture medium and the fibrin-based constructs were cultured statically for a week followed by cyclic distention for another week. The tissues were examined for myogenic differentiation, vascular reactivity, mechanical properties and ECM content. Our results showed that some aspects of vascular function were differentially affected by growth factor presentation vs. pulsatile force application, while others were synergistically enhanced by both. Overall, this two-prong biomimetic approach improved ECM secretion, vascular reactivity and mechanical properties of vascular constructs. These findings may be applied in other tissue engineering applications such as cartilage, tendon or cardiac regeneration where growth factors TGF-ß1 and mechano-stimulation play critical roles.

Engineering fibrin-binding TGF-ß1 for sustained signaling and contractile function of MSC based vascular constructs.

We present a strategy to conjugate TGF-ß1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-ß1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-ß1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-ß1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-ß1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-ß1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-ß1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.