Browsing by Subject "stem cells"

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  • Sanz-Garcia, Andres; Sodupe-Ortega, Enrique; Pernia-Espinoza, Alpha; Shimizu, Tatsuya; Escobedo-Lucea, Carmen (2020)
    Three-dimensional (3D) bioprinting promises to be essential in tissue engineering for solving the rising demand for organs and tissues. Some bioprinters are commercially available, but their impact on the field of Tissue engineering (TE) is still limited due to their cost or difficulty to tune. Herein, we present a low-cost easy-to-build printhead for microextrusion-based bioprinting (MEBB) that can be installed in many desktop 3D printers to transform them into 3D bioprinters. We can extrude bioinks with precise control of print temperature between 2-60 degrees C. We validated the versatility of the printhead, by assembling it in three low-cost open-source desktop 3D printers. Multiple units of the printhead can also be easily put together in a single printer carriage for building a multi-material 3D bioprinter. Print resolution was evaluated by creating representative calibration models at different temperatures using natural hydrogels such as gelatin and alginate, and synthetic ones like poloxamer. Using one of the three modified low-cost 3D printers, we successfully printed cell-laden lattice constructs with cell viabilities higher than 90% after 24-h post printing. Controlling temperature and pressure according to the rheological properties of the bioinks was essential in achieving optimal printability and great cell viability. The cost per unit of our device, which can be used with syringes of different volume, is less expensive than any other commercially available product. These data demonstrate an affordable open-source printhead with the potential to become a reliable alternative to commercial bioprinters for any laboratory.
  • Eskelinen, Eeva-Liisa (2019)
    Autophagy is a conserved catabolic process that delivers cytoplasmic components and organelles to lysosomes for degradation and recycling. This pathway serves to degrade nonfunctional organelles and aggregate-prone proteins, as well as to produce substrates for energy production and biosynthesis. Autophagy is especially important for the maintenance of stem cells, and for the survival and homeostasis of post-mitotic cells like neurons. Functional autophagy promotes longevity in several model organisms. Autophagy regulates immunity and inflammation at several levels and has both anti- and pro-tumorigenic roles in cancer. This review provides a concise overview of autophagy and its importance in cellular and organismal homeostasis, with emphasis on aging, stem cells, neuronal cells, immunity, inflammation, and cancer.
  • Dekoninck, Sophie; Hannezo, Edouard; Sifrim, Alejandro; Miroshnikova, Yekaterina A.; Aragona, Mariaceleste; Malfait, Milan; Gargouri, Souhir; de Neunheuser, Charlotte; Dubois, Christine; Voet, Thierry; Wickström, Sara A.; Simons, Benjamin D.; Blanpain, Cédric (2020)
    Summary During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.
  • Sanz-Navarro, Maria; Delgado, Irene; Torres, Miguel; Mustonen, Tuija; Michon, Frederic; Rice, David P. (2019)
    MEIS1 is a key developmental regulator of several organs and participates in stem cell maintenance in different niches. However, despite the murine continuously growing incisor being a well described model for the study of adult stem cells, Meis1 has not been investigated in a dental context. Here, we uncover that Meis1 expression in the tooth is confined to the epithelial compartment. Its expression arises during morphogenesis and becomes restricted to the mouse incisor epithelial stem cell niche, the labial cervical loop. Meis1 is specifically expressed by Sox2(+) stem cells, which give rise to all dental epithelial cell lineages. Also, we have found that Meis1 in the incisor is coexpressed with potential binding partner Pbx1 during both embryonic and adult stages. Interestingly, Meis2 is present in different areas of the forming tooth and it is not expressed by dental epithelial stem cells, suggesting different roles for these two largely homologous genes. Additionally, we have established the expression patterns of Meis1 and Meis2 during tongue, hair, salivary gland and palate formation. Finally, analysis of Meis1-null allele mice indicated that, similarly, to SOX2, MEIS1 is not essential for tooth initiation, but might have a role during adult incisor renewal.
  • Ilmonen, Lotta (Helsingfors universitet, 2003)
    Stamceller definieras ofta enligt det organ de befinner sig i. Det antas att stamceller från ett visst organ kan ge upphov till alla celler av detta organ och bara till dessa celler. Därför har färska forskningar väckt uppmärksamhet då de påvisat att stamceller kan välja otypiska specialiceringslinjer. Experiment har utförts på bestrålade djur. Det har varit oklart vilken betydelse donatorns stamceller har på mottagarens tillväxt i fysiologiska tillstånd. I denna studie har vi använt kimeriska tvillingkalvar i vilkas nonhematopoetiska vävnader donatorns stamceller har spårats. Med tvillingar av olika kön resulterar placentala anastomoser i tidig embryonal utveckling, i en kimerisk, steril kokalv som kallas freemartin. Med hjälp av Y-kromosom riktad in situ hybridisering kan vi spåra donatorns, tjurkalvens celler i mottagarens, freemartin-kalvens vävnader i deras naturliga omgivning. Hematopoetiska celler spårades med att identifiera deras CD45 yt-antigen och kunde därmed, i kombination med in situ hybridiseringen ekskluderas. Vi har hittat nonhematopoetiska celler härstammande från donatorn i vävnader vars ursprung är långt från varandra, så som entodermet och nerv stängen. Nonhematopoetiska celler som härstammar från donatorn var fåtaliga och de var sporadiskt utspridda i vävnaderna. Detta föreslår att de har ringa betydelse i det fysiologiska uppehållet och i utvecklingen av nötkreaturens vävnader.
  • Li, Hao; Hohenstein, Peter; Kuure, Satu (2021)
    The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature kidney, the embryonic kidney hosts at least three types of lineage-specific stem cells that give rise to (a) a ureter and collecting duct system, (b) nephrons, and (c) mesangial cells together with connective tissue of the stroma. Extensive interest has been raised towards these embryonic progenitor cells, which are normally lost before birth in humans but remain part of the undifferentiated nephrogenic rests in the pediatric renal cancer Wilms tumor. Here, we discuss the current understanding of kidney-specific embryonic progenitor regulation in the innate environment of the developing kidney and the types of disruptions in their balanced regulation that lead to the formation of Wilms tumor.
  • Mihaylova, Maria M.; Cheng, Chia-Wei; Cao, Amanda Q.; Tripathi, Surya; Mana, Miyeko D.; Bauer-Rowe, Khristian E.; Abu-Remaileh, Monther; Clavain, Laura; Erdemir, Aysegul; Lewis, Caroline A.; Freinkman, Elizaveta; Dickey, Audrey S.; La Spada, Albert R.; Huang, Yanmei; Bell, George W.; Deshpande, Vikram; Carmeliet, Peter; Katajisto, Pekka; Sabatini, David M.; Yilmaz, Ömer H. (2018)
    Diet has a profound effect on tissue regeneration in diverse organisms, and low caloric states such as intermittent fasting have beneficial effects on organismal health and age-associated loss of tissue function. The role of adult stem and progenitor cells in responding to short-term fasting and whether such responses improve regeneration are not well studied. Here we show that a 24 hr fast augments intestinal stem cell (ISC) function in young and aged mice by inducing a fatty acid oxidation (FAO) program and that pharmacological activation of this program mimics many effects of fasting. Acute genetic disruption of Cpt1a, the rate-limiting enzyme in FAO, abrogates ISC-enhancing effects of fasting, but long-term Cpt1a deletion decreases ISC numbers and function, implicating a role for FAO in ISC maintenance. These findings highlight a role for FAO in mediating pro-regenerative effects of fasting in intestinal biology, and they may represent a viable strategy for enhancing intestinal regeneration.
  • Nava, Michele M.; Miroshnikova, Yekaterina A.; Biggs, Leah C.; Whitefield, Daniel B.; Metge, Franziska; Boucas, Jorge; Vihinen, Helena; Jokitalo, Eija; Li, Xinping; García Arcos, Juan Manuel; Hoffmann, Bernd; Merkel, Rudolf; Niessen, Carien M.; Dahl, Kris Noel; Wickström, Sara A. (2020)
    Summary Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.
  • Tulokas, Sari (Helsingfors universitet, 2016)
    This study compared proliferation rate and expression of cardiac stem cell markers in one day seven days old mice hearts (1D and 7D). Primary antibodies Ki-67 and C-kit were used in immunohistochemistry and primers C-kit, GATA-4, Myh6, Sca-1 and Mesp1 were used in quantitative PCR. This study found a considerably higher proliferation rate and a higher number of C-kit+ stem cells in 1D than in 7D samples, but the difference was not statistically significant due to a small sample size. A significant difference in the expression of other studied markers was not found. These findings suggest that a decrease in proliferation rate and the number of C-kit+ stem cells coincide with the loss of regenerative potential. Therefore, it is possible that either, or both, of these changes are responsible for the closure of the regenerative window.
  • Ahlnäs-Piña, Karen (Helsingin yliopisto, 2020)
    A robust energy metabolism in humans relies on functional insulin-producing beta cells for maintenance of normoglycemia. Dysfunctional beta cells or malfunctioning insulin is the underlying cause for all forms of diabetes. With current management of different types of diabetes, patients continue to face life-threatening risk of hypoglycemia and other associated pathologies. Research has been challenging due to difficulties in studying beta cells in vitro, as human donor-derived beta cells do not expand well in laboratory conditions. At the time when this study was performed, only a few protocols had been published for beta-cell differentiation. These protocols were often difficult to reproduce in different cell lines and resulted in a low yield of differentiated cells. Both pancreas and liver develop from the same precursor cells called the definitive endoderm (DE). Factors directing the differentiation of definitive endoderm towards a pancreatic endocrine progenitor fate are not fully known. Previous unpublished data indicated the presence of transforming growth factor beta (TGF-β) superfamily inhibitors in the early stages of a developing murine pancreas. In this study, protocols for producing pancreas-specific endoderm cells from human pluripotent stem cells (hPSC) were tested and developed further. The primary aim of this study was to inhibit TGF-β mediated signaling, follow-up and report the effects in definitive endoderm specification. Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) were differentiated into definitive endoderm (DE) using directed differentiation, followed by five experimental conditions with TGF-β inhibitors. Quantitative real-time PCR (q-PCR), flow-cytometry (FC), immunocytochemistry (ICC), and –fluorescence (IF) techniques were used to examine the gene- and protein expression of the cells at specific time points of the protocol. The gene expression levels of known hepatic and pancreatic markers were analyzed and compared between the cell lines. The current DE differentiation protocol, consisting of the DE stage, followed by a four-day culture, showed a downregulation of hepatic markers with and without TGF-β inhibitors. The examined protocols resulted in heterogenic cell populations, consequent to previously reported challenges in beta-cell progenitor differentiation. This study provided valuable information and a platform for further research on the differentiation of pancreatic endocrine progenitor cells.
  • Juuri, Emma; Tikka, Pauli; Domanskyi, Andrii; Corfe, Ian; Morita, Wataru; Mckinnon, Peter J.; Jandova, Nela; Balic, Anamaria (2022)
    Ptch receptors 1 and 2 mediate Hedgehog signaling pivotal for organ development and homeostasis. In contrast to embryonic lethal Ptch1(-/-) phenotype, Ptch2(-/-) mice display no effect on gross phenotype. In this brief report, we provide evidence of changes in the putative incisor mesenchymal stem cell (MSC) niches that contribute to accelerated incisor growth, as well as intriguing changes in the bones and skin which suggest a role for Ptch2 in the regulation of MSCs and their regenerative potential. We employed histological, immunostaining, and computed tomography (mu CT) analyses to analyze morphological differences between Ptch2(-/-) and wild-type incisors, long bones, and skins. In vitro CFU and differentiation assays were used to demonstrate the MSC content and differentiation potential of Ptch2(-/-) bone marrow stromal cells. Wound healing assay was performed in vivo and in vitro on 8-week-old mice to assess the effect of Ptch2 on the wound closure. Loss of Ptch2 causes increases in the number of putative MSCs in the continuously growing incisor, associated with increased vascularization observed in the tooth mesenchyme and the neurovascular bundle. Increased length and volume of Ptch2(-/-) bones is linked with the increased number and augmented in vitro differentiation potential of MSCs in the bone marrow. Dynamic changes in the Ptch2(-/-) skin thickness relate to changes in the mesenchymal compartment and impact the wound closure potential. The effects of Ptch2 abrogation on the postnatal MSCs suggest a crucial role for Ptch2 in Hedgehog signaling regulation of the organ regenerative potential.
  • Sandelin, Amanda (Helsingin yliopisto, 2022)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved protein with pleiotropic therapeutic effects in several disease models, including Parkinson’s disease (PD), diabetes and stroke. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and many GWAS-based genes predisposing for PD are involved in oxidative stress. MANF has been shown to alleviate oxidative stress in PD models, however, the role of MANF in the antioxidant defense and mitochondrial respiration is not fully understood. By performing bulk RNA sequencing on wildtype and MANF knockout (MANF-KO) human embryonic stem cells (hESCs), we uncovered several genes involved in antioxidant defense to be up- or downregulated in MANF-KO hESC. Here we report that MANF-KO hESCs do not express the evolutionary conserved antioxidant enzyme catalase. We show that the loss of catalase makes the MANF-KO hESCs more vulnerable to hydrogen peroxide indued oxidative stress, and that MANF-KO hESCs have a reduced maximal respiration and spare respiratory capacity. Additionally, we examined if the loss of catalase in MANF-KO hESCs inhibits the differentiation of the cells to human dopaminergic neurons in vitro. We show that MANF-KO hESCs differentiate to TH+/MAP2+ cells despite a sustained deficiency of catalase, but the MANF-KO DA cultures tend to have a reduced spare respiratory capacity and higher basal glycolytic activity. To elucidate the structure-to-function relationship of MANF we utilize molecular dynamics simulations in combination with spin relaxation data from nuclear magnetic resonance spectroscopy. By examining the two-domain nature of MANF in different intracellular conditions we provide insight of the biological relevance of MANF interactions. Here we show that MANF conformational ensemble is more compact than previously reported. By simulating MANF in the presence of calcium and ATP, in neutral and low pH, we observed competitive binding of ATP and calcium to MANF. This study provides novel evidence of a regulatory role of MANF in the cellular antioxidant defense and explores the biological relevance of ATP and calcium binding to MANF.
  • Auno, Samuli (Helsingfors universitet, 2019)
    Heart failure is a disease of major social and economic impact. The disease is most commonly onset by extensive cardiomyocyte death following a myocardial infarction. Five-year mortality of heart failure is higher than some cancers. Loss of cardiac muscle tissue leads to pathological thickening and fibrosis of the left ventricular wall, which eventually further diminish cardiac function. Cardiomyocytes hardly proliferate, which also exacerbates the problem. Several cell signalling pathways are indicated in pathological reprogramming of the heart and the exact significance of these pathways remains to be demonstrated. Treatment strategies based on renewing cardiac muscle, such as direct injection of stem cells into the myocardium, have failed to reach clinically significant effects on heart failure patients. Direct inhibition of pathological cardiac reprogramming by using small molecule modulators remains as an auspicious strategy to treat heart failure. GATA4, or GATA binding protein 4, is a transcription factor expressed mainly in heart, lung, intestine, gonad and liver tissues, which regulates tissue renewal and cell proliferation by controlling protein transcription. GATA4 binds to GATA sequences in DNA with two zinc finger moieties and enables transcription of target genes. Interactions of GATA4 and several other transcription factors are in central role of guiding heart development, hypertrophy and fibrosis. One of these transcription factors is NKX2-5, which synergistically interacts with GATA4. Inhibition of this interaction in rat myocardial infarction model has been shown to protect against development of heart failure. A screening campaign against the transcriptional synergy of GATA4 and NKX2-5 found potent small molecule inhibitors of this interaction, but these compounds are characterised with stem cell toxicity. The aim of the study was to design and synthesise novel derivatives of GATA4-NKX2-5 synergy inhibitor hit molecule with reduced stem cell toxicity. Modifications on the phenyl ring of the hit molecule were designed, which either increase electron density of the ring or possibly alter the torsional angle between the phenyl and isoxazole ring moieties. Activity of the compounds was studied on a luciferase reporter gene system in COS-1 cells and toxicity was analysed on IMR90 human induced pluripotent stem cell line. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide and lactate dehydrogenase (LDH) assays were selected to measure toxicity on stem cells. Stem cell toxicity of several previously synthesised compounds was assayed in parallel with the novel derivatives. Ten novel derivatives were designed, synthesised and assayed. Four of the new compounds, a mono-ortho-methyl, a di-ortho-methyl, a di-meta-methoxy and cyclohexyl derivatives were found to be equipotent inhibitors of reporter gene activity compared to the hit compound. Additionally, the mono-ortho-methyl, di-ortho-methyl and di-meta-methoxy derivatives were less toxic to stem cells than the hit molecule in the MTT assay. Several other derivatives were found to be less toxic, but also non-active in the luciferase assay. None of the studied compounds exhibited notable necrotic toxicity on stem cells, as examined by the LDH assay. According to this study it may be concluded that substituents of the hit molecule phenyl ring may be altered to decrease stem cell toxicity of the compound. Some of the alterations, most notably substituents in the para-position of the phenyl ring and substitution of the phenyl ring with smaller saturated hydrocarbon rings, diminish the activity of the hit compound. Remarkable toleration of ortho-substitution reinforces the hypothesis of phenyl-isoxazole torsional angle significance for toxicity. On the other hand, addition of two methoxy groups to both meta positions most likely lacks any substantial effect on the torsional angle, which implies another mechanism of toxicity avoidance. Activity and improved safety of the novel inhibitors should be confirmed in animal models before any decisive conclusions on the effects of structural modifications on the hit molecule can be made. In addition, other mechanisms of toxicity should be studied with relevant cell-based assays.