Browsing by Subject "DIFFERENTIATION"

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  • Beauchamp, Philippe; Jackson, Christopher B.; Ozhathil, Lijo Cherian; Agarkova, Irina; Galindo, Cristi L.; Sawyer, Douglas B.; Suter, Thomas M.; Zuppinger, Christian (2020)
    Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions.Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR).Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture.Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.
  • Radhakrishnan, Dhanya; Shanmukhan, Anju Pallipurath; Kareem, Abdul; Aiyaz, Mohammed; Varapparambathu, Vijina; Toms, Ashna; Kerstens, Merijn; Valsakumar, Devisree; Landge, Amit N.; Shaji, Anil; Mathew, Mathew K.; Sawchuk, Megan G.; Scarpella, Enrico; Krizek, Beth A.; Efroni, Idan; Mähönen, Ari Pekka; Willemsen, Viola; Scheres, Ben; Prasad, Kalika (2020)
    Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.
  • Harma, Ville; Virtanen, Johannes; Mäkelä, Rami; Happonen, Antti; Mpindi, John-Patrick; Knuuttila, Matias; Kohonen, Pekka; Lötjönen, Jyrki; Kallioniemi, Olli; Nees, Matthias (2010)
  • Grubman, Alexandra; Vandekolk, Teresa H.; Schröder, Jan; Sun, Guizhi; Hatwell-Humble, Jessica; Chan, Jonathan; Oksanen, Minna; Lehtonen, Sarka; Hunt, Cameron; Koistinaho, Jan E.; Nilsson, Susan K.; Haynes, John M.; Pouton, Colin W.; Polo, Jose M. (2020)
    Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.
  • 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)
  • Comai, Glenda; Heude, Eglantine; Mella, Sebastian; Paisant, Sylvain; Pala, Francesca; Gallardo, Mirialys; Langa, Francina; Kardon, Gabrielle; Gopalakrishnan, Swetha; Tajbakhsh, Shahragim (2019)
    In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.
  • Hytönen, Marjo K.; Lohi, Hannes (2019)
    Hairlessness is a breed-specific feature selected for in some dog breeds but a rare abnormality in some others such as Scottish Deerhounds (SD). In SDs, the affected puppies are born with sparse hair but lose it within the first 2months leaving the dogs completely hairless. The previous studies have implicated variants in FOXI3 and SGK3 in hairlessness; however, the known variants do not explain hairlessness in all breeds such as SDs. We investigated the genetic cause in 66 SDs, including a litter with two hairless dogs. We utilized a combined approach of genome-wide homozygosity mapping and whole-genome sequencing of a hairless SD followed by recessive filtering according to a recessive model against 340 control genomes. Only two homozygous-coding variants were discovered in the homozygosity regions, including a 1-bp insertion in exon 2 of SGK3. This results in a predicted frameshift and very early truncation (49/490 amino acids) of the SGK3 protein. Additional screening of the recessive variant demonstrated a full segregation with the hairlessness and a 12% carrier frequency in the SD breed. The variant was not found in the related Irish Wolfhound breed. This study identifies the second hairless variant in the SGK3 gene in dogs and further highlights its role as a candidate gene for androgen-independent hair loss or alopecia in human.
  • Khrunin, Andrey V.; Khokhrin, Denis V.; Filippova, Irina N.; Esko, Tonu; Nelis, Mari; Bebyakova, Natalia A.; Bolotova, Natalia L.; Klovins, Janis; Nikitina-Zake, Liene; Rehnström, Karola Hannele; Ripatti, Samuli; Schreiber, Stefan; Franke, Andre; Macek, Milan; Krulisova, Veronika; Lubinski, Jan; Metspalu, Andres; Limborska, Svetlana A. (2013)
  • Leigh, Robert S.; Ruskoaho, Heikki J.; Kaynak, Bogac L. (2020)
    Reliable in vitro models to assess developmental toxicity of drugs and chemicals would lead to improvement in fetal safety and a reduced cost of drug development. The validated embryonic stem cell test (EST) uses cardiac differentiation of mouse embryonic stem cells (mESCs) to predict in vivo developmental toxicity, but does not take into account the stage-specific patterning of progenitor populations into anterior (ventricular) and posterior (atrial) compartments. In this study, we generated a novel dual reporter mESC line with fluorescent reporters under the control of anterior and posterior cardiac promoters. Reporter expression was observed in nascent compartments in transgenic mouse embryos, and mESCs were used to develop differentiation assays in which chemical modulators of Wnt (XAV939: 3, 10 mu M), retinoic acid (all-trans retinoic acid: 0.1, 1, 10 mu M; 9-cis retinoic acid: 0.1, 1, 10 mu M; bexarotene 0.1, 1, 10 mu M), and Tgf-beta (SB431542: 3, 10 mu M) pathways were tested for stage- and dose-dependent effects on in vitro anterior-posterior patterning. Our results suggest that with further development, the inclusion of anterior-posterior reporter expression could be part of a battery of high-throughput tests used to identify and characterize teratogens.
  • Vuoristo, Sanna; Toivonen, Sanna; Weltner, Jere; Mikkola, Milla; Ustinov, Jarkko; Trokovic, Ras; Palgi, Jaan; Lund, Riikka; Tuuri, Timo; Otonkoski, Timo (2013)
  • Narva, Elisa; Stubb, Aki; Guzman, Camilo; Blomqvist, Matias; Balboa, Diego; Lerche, Martina; Saari, Markku; Otonkoski, Timo; Ivaska, Johanna (2017)
    Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure.
  • Sola-Carvajal, Agustin; Revechon, Gwladys; Helgadottir, Hafdis T.; Whisenant, Daniel; Hagblom, Robin; Döhla, Julia; Katajisto, Pekka; Brodin, David; Fagerstrom-Billai, Fredrik; Viceconte, Nikenza; Eriksson, Maria (2019)
    Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/beta-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of beta-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.
  • Ajdary, Rubina; Huan, Siqi; Zanjanizadeh Ezazi, Nazanin; Xiang, Wenchao; Grande, Rafael; Santos, Hélder A.; Rojas, Orlando J. (2019)
    Nanocellulose has been demonstrated as a suitable material for cell culturing, given its similarity to extracellular matrices. Taking advantage of the shear thinning behavior, nanocellulose suits three-dimensional (3D) printing into scaffolds that support cell attachment and proliferation. Here, we propose aqueous suspensions of acetylated nanocellulose of a low degree of substitution for direct ink writing (DM). This benefits from the heterogeneous acetylation of precursor cellulosic fibers, which eases their deconstruction and confers the characteristics required for extrusion in DIW. Accordingly, the morphology of related 3D printed architectures and their performance during drying and rewetting as well as interactions with living cells are compared with those produced from typical unmodified and TEMPO-oxidized nanocelluloses. We find that a significantly lower concentration of acetylated nanofibrils is needed to obtain bioinks of similar performance, affording more porous structures. Together with their high surface charge and axial aspect, acetylated nanocellulose produces dimensionally stable monolithic scaffolds that support drying and rewetting, required for packaging and sterilization. Considering their potential uses in cardiac devices, we discuss the interactions of the scaffolds with cardiac myoblast cells. Attachment, proliferation, and viability for 21 days are demonstrated. Overall, the performance of acetylated nanocellulose bioinks opens the possibility for reliable and scaleup fabrication of scaffolds appropriate for studies on cellular processes and for tissue engineering.
  • Sobral-Leite, Marcelo; Wesseling, Jelle; Smit, Vincent T. H. B. M.; Nevanlinna, Heli; van Miltenburg, Martine H.; Sanders, Joyce; Hofland, Ingrid; Blows, Fiona M.; Coulson, Penny; Patrycja, Gazinska; Schellens, Jan H. M.; Fagerholm, Rainer; Heikkila, Paivi; Aittomaki, Kristiina; Blomqvist, Carl; Provenzano, Elena; Ali, Hamid Raza; Figueroa, Jonine; Sherman, Mark; Lissowska, Jolanta; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Phillips, Kelly-Anne; Couch, Fergus J.; Olson, Janet E.; Vachon, Celine; Visscher, Daniel; Brenner, Hermann; Butterbach, Katja; Arndt, Volker; Holleczek, Bernd; Hooning, Maartje J.; Hollestelle, Antoinette; Martens, John W. M.; van Deurzen, Carolien H. M.; van de Water, Bob; Broeks, Annegien; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Easton, Douglas F.; Pharoah, Paul D. P.; Garcia-Closas, Montserrat; de Graauw, Marjo; Schmidt, Marjanka K.; kConFab AOCS Investigators (2015)
    Background: Annexin A1 (ANXA1) is a protein related with the carcinogenesis process and metastasis formation in many tumors. However, little is known about the prognostic value of ANXA1 in breast cancer. The purpose of this study is to evaluate the association between ANXA1 expression, BRCA1/2 germline carriership, specific tumor subtypes and survival in breast cancer patients. Methods: Clinical-pathological information and follow-up data were collected from nine breast cancer studies from the Breast Cancer Association Consortium (BCAC) (n = 5,752) and from one study of familial breast cancer patients with BRCA1/2 mutations (n = 107). ANXA1 expression was scored based on the percentage of immunohistochemical staining in tumor cells. Survival analyses were performed using a multivariable Cox model. Results: The frequency of ANXA1 positive tumors was higher in familial breast cancer patients with BRCA1/2 mutations than in BCAC patients, with 48.6 % versus 12.4 %, respectively; P <0.0001. ANXA1 was also highly expressed in BCAC tumors that were poorly differentiated, triple negative, EGFR-CK5/6 positive or had developed in patients at a young age. In the first 5 years of follow-up, patients with ANXA1 positive tumors had a worse breast cancer-specific survival (BCSS) than ANXA1 negative (HRadj = 1.35; 95 % CI = 1.05-1.73), but the association weakened after 10 years (HRadj = 1.13; 95 % CI = 0.91-1.40). ANXA1 was a significant independent predictor of survival in HER2+ patients (10-years BCSS: HRadj = 1.70; 95 % CI = 1.17-2.45). Conclusions: ANXA1 is overexpressed in familial breast cancer patients with BRCA1/2 mutations and correlated with poor prognosis features: triple negative and poorly differentiated tumors. ANXA1 might be a biomarker candidate for breast cancer survival prediction in high risk groups such as HER2+ cases.
  • Twardziok, Monika; Schroder, Paul C.; Krusche, Johanna; Casaca, Vera I.; Illi, Sabina; Bock, Andreas; Loss, Georg J.; Kabesch, Michael; Toncheva, Antoaneta A.; Roduit, Caroline; Depner, Martin; Genuneit, Jon; Renz, Harald; Roponen, Marjut; Weber, Juliane; Braun-Fahrlander, Charlotte; Riedler, Josef; Lauener, Roger; Vuitton, Dominique Angele; Dalphin, Jean-Charles; Pekkanen, Juha; von Mutius, Erika; Schaub, Bianca; PASTURE Study Grp; Hyvarinen, Anne; Karvonen, Anne M.; Kirjavainen, Pirkka V.; Remes, Sami; Kaulek, Vincent; Dalphin, Marie-Laure; Ege, Markus; Pfefferle, Petra I.; Doekes, Gert (2017)
    Several studies report an important role of CD8(+) cytotoxic T-cells in atopy. Farm children show protection against atopy development, partly explained by CD4(+) T-cell subtypes. Additional effects of CD8(+) T-cells are unknown being investigated in this study within the PASTURE/EFRAIM birth cohort in PBMCs from farming and non-farming 6-year-old (N = 76) German children. CD3(+) CD8(+) CD25(+) T-cells were analyzed by flow cytometry. Genotyping of 17q21 locus-SNPs associated with childhood asthma was performed. No differences in CD8(+) T-cell subsets were seen between farmers and non-farmers regardless of asthma. Among farm children, asthmatics displayed increased CD3(+) CD8(low)(CD25(+)) T-cells compared to non-asthmatics. Asthmatic farm children exhibited a lower PI-induced stimulatory capacity of CD3(+) CD8(low)(CD25(+)) cells and a lower IFN-gamma secretion than non-asthmatic farm children. Among farm children with GSDMB and ORMDL3 risk alleles, asthmatics displayed higher CD3(+) CD8(low) cells than non-asthmatics. Our data indicates a specific role of CD8(low) T-cells in asthmatic farm children. (C) 2017 Elsevier Inc. All rights reserved.
  • Ramsay, Eva; Ravina, Manuela; Sarkhel, Sanjay; Hehir, Sarah; Cameron, Neil R.; Ilmarinen, Tanja; Skottman, Heli; Kjems, Jrgen; Urtti, Arto; Ruponen, Marika; Subrizi, Astrid (2020)
    Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.
  • Chilov, Dmitri; Sinjushina, Natalia; Saarimäki-Vire, Jonna Marianne; Taketo, Makoto M.; Partanen, Juha (2010)
  • Oswald, Evelyn; Kirschberg, Matthias; Aubin, Francois; Alonso, Angel; Hufbauer, Martin; Akguel, Baki; Auvinen, Eeva (2019)
    Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.