The successful exploitation of human pluripotent stem cells (hPSCs) for research, translational or commercial reasons requires the implementation of a simple and efficient cryopreservation method. Cryopreservation is usually performed with dimethylsulphoxide (DMSO), in addition to animal proteins. However, even at sub-toxic levels, DMSO diminishes the pluripotency capacity of hPSCs and affects their epigenetic system by acting on the three DNA methyltransferases (Dnmts) and histone modification enzymes. Our study aimed to test trehalose-based cryosolutions containing ethylene glycol (EG) or glycerol (GLY) on hESCs RC17, hiPSCs CTR2#6 and long-term neuroepithelial-like stem cells (lt-NES) AF22. Here, we demostrate the effectiveness of these cryosolutions in hPSCs by showing an acceptable rate of cell viability and high stability compared to standard 10% DMSO freezing medium (CS10). All cell lines retained their morphology, self renewal potential and pluripotency, and none of the cryosolutions affected their differentiation potential. Genotoxicity varied among different stem cells types, while trehalose-based cryopreservation did not sensibly alter the homeostasis of endoplasmic reticulum (ER). This study provides evidence that pluripotent and neural stem cells stored in trehalose alone or with other cryoprotectants (CPAs) maintain their functional properties, indicating their potential use in cell therapies if produced in good manufacturing practice (GMP) facility.
Human induced pluripotent stem cell (hiPSC) biobanks are invaluable resources for basic and clinical research, since they provide a sustainable supply of accessible cell lines that meet high quality and safety standards. hiPSCs are particularly useful for understanding disease mechanisms, creating cell models for drug development, and generating novel clinical therapies. For clinical applications and drug discovery, it is fundamental that the acquired pluripotent cell lines never touch animal-derived products nor xenogeneic reagents (Good Manufacturing Practice-grade); whereas for research grade, it is sufficient to operate under Good Laboratory Practice conditions. However, regardless of the end use, it is important that every step in the whole process, starting from the original cells throughout expansion and manipulation, must be performed and recorded rigorously. Here, we describe our biobanking management system that is applied specifically to human pluripotent stem cells.
Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias.
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