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Summary The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy., Highlights • BMSCs cross lineage boundaries toward renal cells via cell-extract reprogramming • Reprogrammed BMSCs acquire proximal tubular-like epithelial cell properties • Reprogrammed BMSCs integrate into proximal tubuli and protect mice from AKI, In this article, Remuzzi, Papadimou, and colleagues show that human BMSCs can be directly reprogrammed into renal proximal tubular-like epithelial cells using epithelial cell extracts. BMSC reprogramming is demonstrated by the acquisition of epithelial features such as morphologic, ultrastructural, antigenic, genetic makeup, and functional properties (organ-forming and renal damage repair capabilities).

Neural stem cells are cells that can self-renew ad infinitum and have the potential to generate immature precursors and mature cells of both neuronal and glial lineages. During recent years, neural stem cells gained attention as major candidates for regenerative and cell replacement therapies in various pathological brain conditions; however, they have recently revealed unforeseen valuable characters. Here the authors review on the state of the field, with a particular focus on the most recent results on the optimisation of neural stem cells isolation/derivation and homogeneous long-term expansion, highlighting advantages of this resource and outlining their potential applications. Bearing in mind that stem-cell based therapies for the diseased and injured brain are still far from being a reality, these cells have been recently opened up to valuable exploitation for disease modelling, drug discovery and toxicology tests as short-term applications.

Huntingtin is a protein that is mutated in Huntington's disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.

Acute kidney injury (AKI) is one of the most relevant health issues, leading to millions of deaths. The magnitude of the phenomenon remarks the urgent need for innovative and effective therapeutic approaches. Cell-based therapy with renal progenitor cells (RPCs) has been proposed as a possible strategy. Studies have shown the feasibility of directing embryonic stem cells or induced Pluripotent Stem Cells (iPSCs) towards nephrogenic intermediate mesoderm and metanephric mesenchyme (MM). However, the functional activity of iPSC-derived RPCs has not been tested in animal models of kidney disease. Here, through an efficient inductive protocol, we directed human iPSCs towards RPCs that robustly engrafted into damaged tubuli and restored renal function and structure in cisplatin-mice with AKI. These results demonstrate that iPSCs are a valuable source of engraftable cells with regenerative activity for kidney disease and create the basis for future applications in stem cell-based therapy.

SummaryThe transcriptional mechanisms underlying lineage specification and differentiation of embryonic stem (ES) cells remain elusive. Oct-3/4 (POU5f1) is one of the earliest transcription factors expressed in the embryo. Both the pluripotency and the fate of ES cells depend upon a tight control of Oct-3/4 expression. We report that transgene- or TGFβ-induced increase in Oct-3/4 mRNA and protein levels in undifferentiated ES cells and at early stages of differentiation triggers expression of mesodermal and cardiac specific genes through Smad2/4. cDNA antisense- and siRNA-mediated inhibition of upregulation of Oct-3/4 in ES cells prevent their specification toward the mesoderm and their differentiation into cardiomyocytes. Similarly, Oct-3/4 siRNA injected in the inner cell mass of blastocysts impairs cardiogenesis in early embryos. Thus, quantitative Oct-3/4 expression is regulated by a morphogen, pointing to a pivotal and physiological function of the POU factor in mesodermal and cardiac commitments of ES cells and of the epiblast.

Ribonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5'-end. We have investigated the effect of synthetic rertinoids (all-trans retinoic acid, acitretin) and arotinoids (Ro 13-7410, Ro 15-0778, Ro, 13-6298 and Ro 15-1570) on RNase P activity isolated for the first time from normal human epidermal keratinocytes (NHEK). All tested compounds but one (Ro 15-1570) revealed a dose-dependent inhibition of RNase P activity, indicating that they may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classic competitive inhibitors. On the basis of the Ki values Ro 13-7410 was found to be the strongest inhibitor among all compounds tested.

For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Micro-computerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and glomeruli. This experimental setup may open the possibility to obtain differentiation of stem cells with long lasting in vitro perfusion.

The effects of two antipsoriatic compounds, calcipotriol and anthralin, separately or in combination on ribonuclease P (RNase P), were investigated using a cell-free system from the slime mold Dictyostelium discoideum . RNase P is an ubiquitous and essential enzyme which endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. The substrate for RNase P assays was an in vitro 32 P-labeled transcript of the Schizosaccharomyces pombe tRNA Ser gene supS1. Enzyme assays were carried out at 37° in 20 μL 50 mM Tris–HCL 7.6 buffer, containing 10 mM NH 4 Cl, 5 mM MgCl 2 , and 10% isopropanol. Calcipotriol or anthralin alone exerted a dose-dependent inhibitory effect on RNase P activity, with the former being more active than the latter in this respect. Simultaneous exposure of the enzyme to both drugs resulted in an enhancement of RNase P inhibition, which was additive. Considering the lack of structural similarities between the substrate (precursor tRNA) of RNase P and the tested drugs, it seems reasonable to suggest that their effects may be due to binding to allosteric inhibition sites of the enzyme. Although our in vitro findings cannot be directly extrapolated to the in vivo human condition, they do suggest that the inhibitory effects of calcipotriol and anthralin on tRNA biogenesis may be implicated in the mechanisms of their antipsoriatic action. Moreover, the additive inhibitory effect of these compounds on RNase P activity provides an experimental basis for their possible combined therapeutic application in the management of psoriasis.

The effects of cholesterol, 7-dehydrocholesterol, vitamin D3 and several synthetic vitamin D3 analogs on ribonuclease P (RNase P) were investigated using a cell-free system from the slime mold Dictyostelium discoideum. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. Among the compounds tested, only calcipotriol was capable of affecting RNase P activity, and revealed a bimodal action at the kinetic phase of the reaction. Depending on the concentration of the drug, both activation and inhibition of tRNA maturation were observed, indicating that calcipotriol may have a direct effect on tRNA biogenesis, possibly associated with the presence of a highly reactive small ring on the side chain of its molecule.

The effect of five different anthralin concentrations on tRNA biogenesis was investi- gated employing the ribonuclease P (RNase P) of the slime mold Dictyostelium discoideum as an in vitro cell-free experimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. Anthralin revealed a dose-dependent inhibition of RNase P activity indicating that this compound may have a direct effect on tRNA biogenesis. Taking into account that anthralin has no structural similarities to the substrate (pre-tRNA) of RNase P, it seems reasonable to suggest that this compound may bind to allosteric inhibition sites of the enzyme.

SHC genes codify for a family of adaptor molecules comprising four genes. Previous data have implicated the Shc(s) molecules in stem cell division and differentiation. Specifically, the p66(ShcA) isoform has been found to contribute to longevity and resistance from oxidative stress. Here we report that p66(ShcA) is up-regulated during in vitro neural induction in embryonic stem cells. p66(ShcA) over-expression in ES cells reduces GSK-3beta kinase activation and increases beta-catenin stabilization and its transcriptional activity. p66(ShcA) over-expression results in ES cells undergoing an anticipated neural induction and accelerated neuronal differentiation. Similar effects are obtained in human ES cells over-expressing p66(ShcA). This study reveals a role for p66(ShcA) in the modulation of Wnt/beta-catenin pathway and in ES cell neuralization which is consistent between mouse and human.

Novel mono- and diacylated spermines, readily obtained using isolable succinimidyl active esters of acidic retinoids for the selective acylation of free spermine or in situ activated acidic retinoids for acylating selectively protected spermine followed by deprotection, were shown to inhibit the ribozyme ribonuclease P more strongly than the parent retinoids.

Embryonic stem (ES) cells represent a source for cell-based regenerative therapies of heart failure. The pluripotency and the plasticity of ES cells allow them to be committed to a cardiac lineage following treatment with growth factors of the transforming growth factor (TGF)-beta superfamily. We describe a protocol designed to turn on expression of cardiac-specific genes in undifferentiated murine ES cells stimulated with BMP2 and/or TGF-beta. Cell commitment results in a significant improvement in spontaneous cardiac differentiation of ES cells both in vitro and in vivo.

The molecular mechanisms governing early cardiogenesis are still largely unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell cycle, has recently emerged as a new candidate regulating cell differentiation. Rb−/− mice die at midgestation and mice lacking E2f1/E2f3, downstream components of the Rb-dependent transcriptional pathway, die of heart failure. To gain insight into the function of Rb pathway in early cardiogenesis, we used Rb−/− embryonic stem (ES) cells differentiating into cardiomyocytes. Rb−/− cells displayed a dramatic delay in expression of cardiac-specific transcription factors and in turn in the whole process of cardiac differentiation. The phenotype of Rb−/− ES cell-derived cardiomyocytes was rescued by reintroducing Rb in cardiac progenitors, by stimulating the BMP-dependent cardiogenic pathway or by overexpression of Nkx2.5. ES cells deficient in the recently identified factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific disruption of Rb–LEK1 interaction into the nucleus of differentiating ES cells recapitulated the delay in cardiac differentiation of Rb−/− ES cells. Thus, we provide evidence for a novel Rb/LEK1-dependent and BMP-independent transcriptional program, which plays a pivotal role in priming ES cells toward a cardiac fate.

The ever-growing resistance of pathogens to antibiotics and the lack of potent antibacterial drugs constitute major problems in the treatment of infectious diseases. Thus, the better understanding of the mode of action of antibiotics at the molecular level is of essential importance. Accumulating evidence points towards RNA as being a crucial target of antibacterial and antiviral drugs. Interestingly, aminoglycosides, one of the most important families of antibiotics, apart from their inhibitory effect on ribosome function, reportedly interfere with various RNA molecules and in vitro suppress the proliferation of human keratinocytes. In this study we investigated the effect of the aminoglycosides neomycin B, paromomycin, tobramycin and gentamycin on ribonuclease P activity from normal human epidermal keratinocytes. All aminoglycosides tested revealed a dose-dependent inhibition of tRNA maturation, which was reduced by increasing Mg2+ ion concentrations, indicating competition of the cationic aminoglycosides with magnesium ions required for catalysis. Our in vitro findings suggest that the inhibitory effects of aminoglycosides on tRNA processing may be implicated in the mechanisms of their antiproliferative action on human epidermal keratinocytes.

The effects of five arotinoids (Ro 13-7410, Ro 15-0778, Ro 15-1570, Ro 13-6298, Ro 40-8757) on ribonuclease P (RNase P) activity were studied in a cell-free system derived from Dictyostelium discoideum. RNase P is a ribonucleoprotein that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. Kinetic analysis showed that these compounds behave as classical competitive inhibitors with Ki values 4.35, 3.6, 2.8 and 0.045 mM for Ro 13-6298, Ro 15-1570, Ro 15-0778 and Ro 13-7410, respectively. Ro 13-7410 was 62, 80 and 97 times more potent in inhibiting the enzyme activity as compared to Ro 15-0778, Ro 15-1570 and Ro 13-6298, respectively, whereas Ro 40-8757 showed no effect on RNase P activity. These results project the significance of the acidic polar terminus in the arotinoid molecule binding to the enzyme. The kinetics of inhibition reflects allosteric interactions of arotinoids with D. discoideum RNase P. Moerover, our findings indicate that the inhibitory effects of arotinoids on tRNA biogenesis can be mediated through mechanisms not involving the retinoid nuclear receptors.

The effect of two naturally occurring (retinol and all-trans retinoic acid) and two synthetic (isotretinoin and acitretin) analogs of vitamin A (retinoids) on tRNA biogenesis was investigated employing the RNase P of Dictyostelium discoideum as an in vitro experimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classical competitive inhibitors. The Ki values determined were 1475 microM for retinol, 15 microM for all-trans retinoic acid, 20 microM for isotretinoin, and 8.0 microM for acitretin. On the basis of these values acitretin is a 184, 2.5, and 1.9 times more potent inhibitor, as compared with retinol, isotretinoin, and all-trans retinoic acid, respectively. Taking into account that retinoids share no structural similarities to precursor tRNA, it is suggested that their kinetic behavior reflects allosteric interactions of these compounds with hydrophobic site(s) of D. discoideum RNase P.