Browsing by Subject "HUMAN FIBROBLASTS"

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  • Papaevgeniou, Nikoletta; Sakellari, Marianthi; Jha, Sweta; Tavernarakis, Nektarios; Holmberg, Carina I.; Gonos, Efstathios S.; Chondrogianni, Niki (2016)
    Aims: Proteasomes are constituents of the cellular proteolytic networks that maintain protein homeostasis through regulated proteolysis of normal and abnormal (in any way) proteins. Genetically mediated proteasome activation in multicellular organisms has been shown to promote longevity and to exert protein antiaggregation activity. In this study, we investigate whether compound-mediated proteasome activation is feasible in a multicellular organism and we dissect the effects of such approach in aging and Alzheimer's disease (AD) progression. Results: Feeding of wild-type Caenorhabditis elegans with 18 alpha-glycyrrhetinic acid (18 alpha-GA; a previously shown proteasome activator in cell culture) results in enhanced levels of proteasome activities that lead to a skinhead-1- and proteasomeactivation-dependent life span extension. The elevated proteasome function confers lower paralysis rates in various AD nematode models accompanied by decreased A beta deposits, thus ultimately decelerating the progression of AD phenotype. More importantly, similar positive results are also delivered when human and murine cells of nervous origin are subjected to 18 alpha-GA treatment. Innovation: This is the first report of the use of 18 alpha-GA, a diet-derived compound as prolongevity and antiaggregation factor in the context of a multicellular organism. Conclusion: Our results suggest that proteasome activation with downstream positive outcomes on aging and AD, an aggregation-related disease, is feasible in a nongenetic manipulation manner in a multicellular organism. Moreover, they unveil the need for identification of antiaging and antiamyloidogenic compounds among the nutrients found in our normal diet.
  • Rajala, Kristiina; Lindroos, Bettina; Hussein, Samer M.; Lappalainen, Riikka S.; Pekkanen-Mattila, Mari; Inzunza, Jose; Rozell, Bjorn; Miettinen, Susanna; Narkilahti, Susanna; Kerkela, Erja; Aalto-Setälä, Katriina; Otonkoski, Timo; Suuronen, Riitta; Hovatta, Outi; Skottman, Heli (2010)
  • Oja, S.; Komulainen, P.; Penttilä, A.; Nystedt, J.; Korhonen, M. (2018)
    Background: Senescent cells are undesirable in cell therapy products due to reduced therapeutic activity and risk of aberrant cellular effects, and methods for assessing senescence are needed. Early-passage mesenchymal stromal cells (MSCs) are known to be small and spindle-shaped but become enlarged upon cell aging. Indeed, cell morphology is routinely evaluated during MSC production using subjective methods. We have therefore explored the possibility of utilizing automated imaging-based analysis of cell morphology in clinical cell manufacturing. Methods: An imaging system was adopted for analyzing changes in cell morphology of bone marrow-derived MSCs during long-term culture. Cells taken from the cultures at the desired passages were plated at low density for imaging, representing morphological changes observed in the clinical-grade cultures. The manifestations of aging and onset of senescence were monitored by population doubling numbers, expression of p16(INK4)a and p21(Cip1/Waf1), beta-galactosidase activity, and telomeric terminal restriction fragment analysis. Results: Cell area was the most statistically significant and practical parameter for describing morphological changes, correlating with biochemical senescence markers. MSCs from passages 1 (p1) and 3 (p3) were remarkably uniform in size, with cell areas between 1800 and 2500 mu m(2). At p5 the cells began to enlarge resulting in a 4.8-fold increase at p6-9 as compared to p1. The expression of p16(INK4a) and activity of beta-galactosidase had a strong correlation with the increase in cell area, whereas the expression of p21(Cip1/Waf1) reached its maximum at the onset of growth arrest and subsequently decreased. Mean telomere length shortened at an apparently constant rate during culture, from 8.2 +/- 0.3 kbp at p1, reaching 6.08 +/- 0.6 kbp at senescence. Conclusions: Imaging analysis of cell morphology is a useful tool for evaluating aging in cell cultures throughout the lifespan of MSCs. Our findings suggest that imaging analysis can reproducibly detect aging-related changes in cell morphology in MSC cultures. These findings suggest that cell morphology is still a supreme measure of cell quality and may be utilized to develop new noninvasive imaging-based methods to screen and quantitate aging in clinical-grade cell cultures.
  • Trokovic, Ras; Weltner, Jere; Noisa, Parinya; Raivio, Taneli; Otonkoski, Timo (2015)
    Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time. (C) 2015 The Authors. Published by Elsevier B.V.
  • Sahu, Biswajyoti; Pihlajamaa, Päivi; Zhang, Kaiyang; Palin, Kimmo; Ahonen, Saija; Cervera, Alejandra; Ristimäki, Ari; Aaltonen, Lauri A.; Hautaniemi, Sampsa; Taipale, Jussi (2021)
    Cancer is the most complex genetic disease known, with mutations implicated in more than 250 genes. However, it is still elusive which specific mutations found in human patients lead to tumorigenesis. Here we show that a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT, MYC) induces senescence in human fibroblasts and primary hepatocytes. However, reprogramming fibroblasts to a liver progenitor fate, induced hepatocytes (iHeps), makes them sensitive to transformation by the same oncogenes. The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. These results show that tumorigenesis is triggered by a combination of three elements: the set of driver mutations, the cellular lineage, and the state of differentiation of the cells along the lineage. Our results provide direct support for the role of cell identity as a key determinant in transformation and establish a paradigm for studying the dynamic role of oncogenic drivers in human tumorigenesis.
  • Laos, M.; Sulg, M.; Herranen, A.; Anttonen, T.; Pirvola, U. (2017)
    p53 is a key component of a signaling network that protects cells against various stresses. As excess p53 is detrimental to cells, its levels are tightly controlled by several mechanisms. The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53. The significance of balanced p53 levels in normal tissues, at different stages of lifetime, is poorly understood. We have studied in vivo how the disruption of Mdm2/p53 interaction affects the early-embryonic otic progenitor cells and their descendants, the auditory supporting cells and hair cells. We found that p53 accumulation, as a consequence of Mdm2 abrogation, is lethal to both proliferative progenitors and non-proliferating, differentiating cells. The sensitivity of postmitotic supporting cells to excess p53 decreases along maturation, suggesting that maturation-related mechanisms limit p53's transcriptional activity towards pro-apoptotic factors. We have also investigated in vitro whether p53 restricts supporting cell's regenerative capacity. Unlike in several other regenerative cellular models, p53 inactivation did not alter supporting cell's proliferative quiescence nor transdifferentiation capacity. Altogether, the postmitotic status of developing hair cells and supporting cells does not confer protection against the detrimental effects of p53 upregulation. These findings might be linked to auditory disturbances observed in developmental syndromes with inappropriate p53 upregulation.
  • De Gregorio, Roberto; Pulcrano, Salvatore; De Sanctis, Claudia; Volpicelli, Floriana; Guatteo, Ezia; von Oerthel, Lars; Latagliata, Emanuele Claudio; Esposito, Roberta; Piscitelli, Rosa Maria; Perrone-Capano, Carla; Costa, Valerio; Greco, Dario; Puglisi-Allegra, Stefano; Smidt, Marten P.; di Porzio, Umberto; Caiazzo, Massimiliano; Mercuri, Nicola Biagio; Li, Meng; Bellenchi, Gian Carlo (2018)
    The differentiation of dopaminergic neurons requires concerted action of morphogens and transcription factors acting in a precise and well-defined time window. Very little is known about the potential role of microRNA in these events. By performing a microRNA-mRNA paired microarray screening, we identified miR-34b/c among the most upregulated microRNAs during dopaminergic differentiation. Interestingly, miR-34b/c modulates Wnt1 expression, promotes cell cycle exit, and induces dopaminergic differentiation. When combined with transcription factors ASCL1 and NURR1, miR-34b/c doubled the yield of transdifferentiated fibroblasts into dopaminergic neurons. Induced dopaminergic (iDA) cells synthesize dopamine and show spontaneous electrical activity, reversibly blocked by tetrodotoxin, consistent with the electrophysiological properties featured by brain dopaminergic neurons. Our findings point to a role for miR-34b/c in neuronal commitment and highlight the potential of exploiting its synergy with key transcription factors in enhancing in vitro generation of dopaminergic neurons.
  • Martin-Lopez, Marta; Maeso-Alonso, Laura; Fuertes-Alvarez, Sandra; Balboa, Diego; Rodriguez-Cortez, Virginia; Weltner, Jere; Diez-Prieto, Inmaculada; Davis, Andrew; Wu, Yaning; Otonkoski, Timo; Flores, Elsa R.; Menendez, Pablo; Marques, Margarita M.; Marin, Maria C. (2017)
    The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming.