Impact of AMPK on cervical carcinoma progression and metastasis.

Cervical cancer (CC) is the fourth most common malignant neoplasm among women. Late diagnosis is directly associated with the incidence of metastatic disease and remarkably limits the effectiveness of conventional anticancer therapies at the advanced tumor stage. In this study, we investigated the role of 5'AMP-activated kinase (AMPK) in the metastatic progression of cervical cancer. Since the epithelial mesenchymal transition (EMT) is known as major mechanism enabling cancer cell metastasis, cell lines, which accurately represent this process, have been used as a research model. We used C-4I and HTB-35 cervical cancer cell lines representing distant stages of the disease, in which we genetically modified the expression of the AMPK catalytic subunit α. We have shown that tumor progression leads to metabolic deregulation which results in reduced expression and activity of AMPK. We also demonstrated that AMPK is related to the ability of cells to acquire invasive phenotype and potential for in vivo metastases, and its activity may inhibit these processes. Our findings support the hypothesis that AMPK is a promising therapeutic target and modulation of its expression and activity may improve the efficacy of cervical cancer treatment.

Nuclear magnetic resonance footprint of Wharton Jelly mesenchymal stem cells death mechanisms and distinctive in-cell biophysical properties in vitro.

The importance of the biophysical characterization of mesenchymal stem cells (MSCs) was recently pointed out for supporting the development of MSC-based therapies. Among others, tracking MSCs in vivo and a quantitative characterization of their regenerative impact by nuclear magnetic resonance (NMR) demands a full description of MSCs' MR properties. In the work, Wharton Jelly MSCs are characterized in a low magnetic field (LF) in vitro by using different approaches. They encompass various settings: MSCs cultured in a Petri dish and cell suspensions; experiments- 1D-T 1 , 1D-T 2 , 1D diffusion, 2D T 1 -T 2 and D-T 2 ; devices- with a bore aperture and single-sided one. Complex NMR analysis with the aid of random walk simulations allows the determination of MSCs T 1 and T 2 relaxation times, cells and nuclei sizes, self-diffusion coefficients of the nucleus and cytoplasm. In addition, the influence of a single layer of cells on the effective diffusion coefficient of water is detected with the application of a single-sided NMR device. It also enables the identification of apoptotic and necrotic cell death and changed diffusional properties of cells suspension caused by compressing forces induced by the subsequent cell layers. The study delivers MSCs-specific MR parameters that may help tracking MSCs in vivo.

Highly Effective Protocol for Differentiation of Induced Pluripotent Stem Cells (iPS) into Melanin-Producing Cells.

Melanin is a black/brown pigment present in abundance in human skin. Its main function is photo-protection of underlying tissues from harmful UV light. Natural sources of isolated human melanin are limited; thus, in vitro cultures of human cells may be a promising source of human melanin. Here, we present an innovative in vitro differentiation protocol of induced pluripotent stem cells (iPS) into melanin-producing cells, delivering highly pigmented cells in quantity and quality incomparably higher than any other methods previously described. Pigmented cells constitute over 90% of a terminally differentiated population and exhibit features characteristic for melanocytes, i.e., expression of specific markers such as MITF-M (microphthalmia-associated transcription factor isoform M), TRP-1 (tyrosinase-related protein 1), and TYR (tyrosinase) and accumulation of black pigment in organelles closely resembling melanosomes. Black pigment is unambiguously identified as melanin with features corresponding to those of melanin produced by typical melanocytes. The advantage of our method is that it does not require any sophisticated procedures and can be conducted in standard laboratory conditions. Moreover, our protocol is highly reproducible and optimized to generate high-purity melanin-producing cells from iPS cells; thus, it can serve as an unlimited source of human melanin for modeling human skin diseases. We speculate that FGF-8 might play an important role during differentiation processes toward pigmented cells.

Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons.

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

Mesenchymal stem cells as a multimodal treatment for nervous system diseases.

Neurological disorders are a massive challenge for modern medicine. Apart from the fact that this group of diseases is the second leading cause of death worldwide, the majority of patients have no access to any possible effective and standardized treatment after being diagnosed, leaving them and their families helpless. This is the reason why such great emphasis is being placed on the development of new, more effective methods to treat neurological patients. Regenerative medicine opens new therapeutic approaches in neurology, including the use of cell-based therapies. In this review, we focus on summarizing one of the cell sources that can be applied as a multimodal treatment tool to overcome the complex issue of neurodegeneration-mesenchymal stem cells (MSCs). Apart from the highly proven safety of this approach, beneficial effects connected to this type of treatment have been observed. This review presents modes of action of MSCs, explained on the basis of data from vast in vitro and preclinical studies, and we summarize the effects of using these cells in clinical trial settings. Finally, we stress what improvements have already been made to clarify the exact mechanism of MSCs action, and we discuss potential ways to improve the introduction of MSC-based therapies in clinics. In summary, we propose that more insightful and methodical optimization, by combining careful preparation and administration, can enable use of multimodal MSCs as an effective, tailored cell therapy suited to specific neurological disorders.

Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson's Disease.

Organoids are becoming particularly popular in modeling diseases that are difficult to reproduce in animals, due to anatomical differences in the structure of a given organ. Thus, they are a bridge between the in vitro and in vivo models. Human midbrain is one of the structures that is currently being intensively reproduced in organoids for modeling Parkinson's disease (PD). Thanks to three-dimensional (3D) architecture and the use of induced pluripotent stem cells (iPSCs) differentiation into organoids, it has been possible to recapitulate a complicated network of dopaminergic neurons. In this work, we present the first organoid model for an idiopathic form of PD. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and patients with the idiopathic form of PD by transduction with Sendai viral vector. iPSCs were differentiated into a large multicellular organoid-like structure. The mature organoids displayed expression of neuronal early and late markers. Interestingly, we observed statistical differences in the expression levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patient and healthy volunteer. The obtained results show immense potential for the application of 3D human organoids in studying the neurodegenerative disease and modeling cellular interactions within the human brain.

Regenerative Potential of the Product "CardioCell" Derived from the Wharton's Jelly Mesenchymal Stem Cells for Treating Hindlimb Ischemia.

In recent years, mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality in regenerative medicine. They hold great promise for treating civilization-wide diseases, including cardiovascular diseases, such as acute myocardial infarction and critical limb ischemia. MSCs isolated from Wharton's jelly (WJ-MSCs) may be utilized in both cell-based therapy and vascular graft engineering to restore vascular function, thereby providing therapeutic benefits for patients. The efficacy of WJ-MSCs lies in their multipotent differentiation ability toward vascular smooth muscle cells, endothelial cells and other cell types, as well as their capacity to secrete various trophic factors, which are potent in promoting angiogenesis, inhibiting apoptosis and modulating immunoreaction. Ischemic limb disease is caused by insufficient nutrient and oxygen supplies resulting from damaged peripheral arteries. The lack of nutrients and oxygen causes severe tissue damage in the limb, thereby resulting in severe morbidities and mortality. The therapeutic effects of the conventional treatments are still not sufficient. Cell transplantations in small animal model (mice) are vital for deciphering the mechanisms of MSCs' action in muscle regeneration. The stimulation of angiogenesis is a promising strategy for the treatment of ischemic limbs, restoring blood supply for the ischemic region. In the present study, we focus on the therapeutic properties of the human WJ-MSCs derived product, Cardio. We investigated the role of CardioCell in promoting angiogenesis and relieving hindlimb ischemia. Our results confirm the healing effect of CardioCell and strongly support the use of the WJ-MSCs in regenerative medicine.

Molecular and Functional Verification of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) Pluripotency.

The properties of mesenchymal stem cells (MSCs), especially their self-renewal and ability to differentiate into different cell lines, are widely discussed. Considering the fact that MSCs isolated from perinatal tissues reveal higher differentiation capacity than most adult MSCs, we examined mesenchymal stem cells isolated from Wharton's jelly of umbilical cord (WJ-MSCs) in terms of pluripotency markers expression. Our studies showed that WJ-MSCs express some pluripotency markers-such as NANOG, OCT-4, and SSEA-4-but in comparison to iPS cells expression level is significantly lower. The level of expression can be raised under hypoxic conditions. Despite their high proliferation potential and ability to differentiate into different cells type, WJ-MSCs do not form tumors in vivo, the major caveat of iPS cells. Owing to their biological properties, high plasticity, proliferation capacity, and ease of isolation and culture, WJ-MSCs are turning out to be a promising tool of modern regenerative medicine.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Concise Review: Mesenchymal Stem Cells in Cardiovascular Regeneration: Emerging Research Directions and Clinical Applications.

Experimental and early clinical data suggest that, due to several unique properties, mesenchymal stem cells (MSCs) may be more effective than other cell types for diseases that are difficult to treat or untreatable. Owing to their ease of isolation and culture as well as their secretory and immunomodulatory abilities, MSCs are the most promising option in the field of cell-based therapies. Although MSCs from various sources share several common characteristics, they also exhibit several important differences. These variations may reflect, in part, specific regional properties of the niches from which the cells originate. Moreover, morphological and functional features of MSCs are susceptible to variations across isolation protocols and cell culture conditions. These observations suggest that careful preparation of manufacturing protocols will be necessary for the most efficient use of MSCs in future clinical trials. A typical human myocardial infarct involves the loss of approximately 1 billion cardiomyocytes and 2-3 billion other (mostly endothelial) myocardial cells, leading (despite maximized medical therapy) to a significant negative impact on the length and quality of life. Despite more than a decade of intensive research, search for the "best" (safe and maximally effective) cell type to drive myocardial regeneration continues. In this review, we summarize information about the most important features of MSCs and recent discoveries in the field of MSCs research, and describe current data from preclinical and early clinical studies on the use of MSCs in cardiovascular regeneration. Stem Cells Translational Medicine 2017;6:1859-1867.

Suicide gene therapy of rhabdomyosarcoma.

Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and young adulthood. Conventional treatment consisting of surgery, chemotherapy and radiotherapy can be insufficient, as long-term survival chances decrease dramatically when cancer recurrence occurs. Due to this fact, efficient treatment of this cancer is still a demanding issue, thus, novel and innovative therapies have to be considered as a part of combined treatment. In the present study, we present effective suicide gene therapy of rhabdomyosarcoma cell line Rh30 involving herpes simplex thymidine kinase (HSV-TK) and ganciclovir (GCV). Transduction of rhabdomyosarcoma cells using lentiviral vectors allowed efficient introduction of HSV-TK gene. In this study we proved high susceptibility of modified cells to ganciclovir resulting in eradication of cancer cells both in vitro and in vivo. Our data revealed strong gap junctional intercellular communication in examined cell line responsible for elimination of unmodified cells by bystander effect, even if HSV-TK-expressing cells comprise only 20% of cultured cells. Moreover, investigated approach is also efficient in vivo, where complete remission of tumors upon only 14 days of systemic administration of GCV can be observed. Obtained results suggest that HSV-TK suicide gene therapy is very promising concept in future clinical studies concerning rhabdomyosarcoma.

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

Antioxidant and anticancer activities of Chenopodium quinoa leaves extracts - in vitro study.

The nutraceutical potential of Chenopodium quinoa Leaves (ChL) was assessed through analyses of their phenolic content, elucidation of the effect of ChL phenolic compounds on cancer cell properties and estimation of their antioxidative activity, bioaccessibility and bioavailability in vitro. Considerable amounts of ferulic, sinapinic and gallic acids, kaempferol, isorhamnetin and rutin were observed in the chemical ChL extract and were linked with its inhibitory effect on prostate cancer cell proliferation, motility and cellular competence for gap junctional communication. Both extracts, chemical and obtained after simulated digestion, exerted an inhibitory effect on lipoxygenase activity, paralleled by their considerable chelating, antioxidative, antiradical and reducing power. These observations indicate that phenolic ChL compounds may exert a chemopreventive and anticarcinogenic effect on oxidative stress and ROS-dependent intracellular signaling via synergic effects. The relatively high potential bioaccessibility and bioavailability of the compounds probably responsible for these effects demonstrates the suitability of ChL for dietary supplementation.