ABSTRACT
APDS2 is caused by mutations in PIK3R1 gene resulting in constitutive PI3Kδ activation. PI3Kδ is predominantly expressed in leukocytes and plays critical roles in regulating immune responses. Here we first derived fibroblast primary cells from a skin biopsy of a patient carrying a heterozygous single T deletion in intron 11 of the PIK3R1 gene. We next present the derivation of an induced pluripotent stem cell (iPS) line using a non-integrative reprogramming technology. Pluripotent-related hallmarks are further shown, including: iPSCs self-renewal and expression of pluripotent and differentiation markers after in vitro differentiation towards embryonic germ layers, assessed by RT-PCR and immunofluorescence.
Subject(s)
Cell Line , Induced Pluripotent Stem Cells , Primary Immunodeficiency Diseases/genetics , Cell Differentiation , Class I Phosphatidylinositol 3-Kinases/genetics , Fibroblasts , Humans , MutationABSTRACT
Bioartificial lungs re-engineered from decellularized organ scaffolds are a promising alternative to lung transplantation. Critical features for improving scaffold repopulation depend on the mechanical properties of the cell microenvironment. However, the mechanics of the lung extracellular matrix (ECM) is poorly defined. The local mechanical properties of the ECM were measured in different regions of decellularized rat lung scaffolds with atomic force microscopy. Lungs excised from rats (n=11) were decellularized with sodium dodecyl sulfate (SDS) and cut into ~7µm thick slices. The complex elastic modulus (G(∗)) of lung ECM was measured over a frequency band ranging from 0.1 to 11.45Hz. Measurements were taken in alveolar wall segments, alveolar wall junctions and pleural regions. The storage modulus (G', real part of G(∗)) of alveolar ECM was ~6kPa, showing small changes between wall segments and junctions. Pleural regions were threefold stiffer than alveolar walls. G' of alveolar walls and pleura increased with frequency as a weak power law with exponent 0.05. The loss modulus (Gâ³, imaginary part of G(∗)) was 10-fold lower and showed a frequency dependence similar to that of G' at low frequencies (0.1-1Hz), but increased more markedly at higher frequencies. Local differences in mechanical properties and topology of the parenchymal site could be relevant mechanical cues for regulating the spatial distribution, differentiation and function of lung cells.
Subject(s)
Cellular Microenvironment/physiology , Extracellular Matrix/physiology , Extracellular Matrix/ultrastructure , Lung/physiology , Lung/ultrastructure , Microscopy, Atomic Force/methods , Tissue Scaffolds , Animals , Bioprosthesis , Cell-Free System/physiology , Elastic Modulus/physiology , Equipment Failure Analysis , Hardness , Materials Testing , Rats , Rats, Sprague-DawleyABSTRACT
Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.
Subject(s)
Induced Pluripotent Stem Cells/physiology , Meiosis , 3-Hydroxysteroid Dehydrogenases/metabolism , Adaptor Proteins, Signal Transducing , Antigens, CD/metabolism , Benzothiazoles/pharmacology , Cell Culture Techniques , Cell Cycle Proteins , Cell Differentiation/drug effects , Cell Line , Colforsin/pharmacology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA Methylation , DNA-Binding Proteins , Fibroblast Growth Factor 2/pharmacology , Gene Expression , Gene Expression Regulation , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Intermediate Filament Proteins/metabolism , Karyotyping , Leukemia Inhibitory Factor/pharmacology , Male , Nerve Tissue Proteins/metabolism , Nestin , Nuclear Proteins/metabolism , Ploidies , Promoter Regions, Genetic , Proteins/genetics , Proteins/metabolism , Spermatogonia/cytology , Spermatogonia/metabolism , Stage-Specific Embryonic Antigens/metabolism , Triazoles/pharmacology , Vimentin/metabolismABSTRACT
A plasma process for the surface modification of HA powders has been developed. Acrylic acid and acrylic acid/octadiene plasma deposited films onto HA particles have demonstrated to interact with SBF allowing the calcium dissolution-precipitation mechanism. Therefore, a nanostructured composite between HA and a self-assembling peptide scaffold (RAD16-I) has been developed. The differentiation of mESC in this scaffold has been studied, in order to test the osteogenic capacity of the new composite material. We have observed that the mESC can be induced to produce Ca salts (mineralization) in a 3D-microenvironment and moreover, this activity can be enhanced by the presence of HA particles into the nanofiber scaffold.
