Transgene-Free Cynomolgus Monkey iPSCs Generated under Chemically Defined Conditions.

Non-human primates (NHPs) are pivotal animal models for translating novel cell replacement therapies into clinical applications, including validating the safety and efficacy of induced pluripotent stem cell (iPSC)-derived products. Preclinical development and the testing of cell-based therapies ideally comprise xenogeneic (human stem cells into NHPs) and allogenic (NHP stem cells into NHPs) transplantation studies. For the allogeneic approach, it is necessary to generate NHP-iPSCs with generally equivalent quality to the human counterparts that will be used later on in patients. Here, we report the generation and characterization of transgene- and feeder-free cynomolgus monkey (Macaca fascicularis) iPSCs (Cyno-iPSCs). These novel cell lines have been generated according to a previously developed protocol for the generation of rhesus macaque, baboon, and human iPSC lines. Beyond their generation, we demonstrate the potential of the novel Cyno-iPSCs to differentiate into two clinically relevant cell types, i.e., cardiomyocytes and neurons. Overall, we provide a resource of novel iPSCs from the most frequently used NHP species in the regulatory testing of biologics and classical pharmaceutics to expand our panel of iPSC lines from NHP species with high relevance in preclinical testing and translational research.

Generation of marmoset primordial germ cell-like cells under chemically defined conditions.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and oocytes, which transmit genetic/epigenetic information across generations. Mouse PGC and subsequent gamete development can be fully reconstituted in vitro, opening up new avenues for germ cell studies in biomedical research. However, PGCs show molecular differences between rodents and humans. Therefore, to establish an in vitro system that is closely related to humans, we studied PGC development in vivo and in vitro in the common marmoset monkey Callithrix jacchus (cj). Gonadal cjPGCs at embryonic day 74 express SOX17, AP2Ɣ, BLIMP1, NANOG, and OCT4A, which is reminiscent of human PGCs. We established transgene-free induced pluripotent stem cell (cjiPSC) lines from foetal and postnatal fibroblasts. These cjiPSCs, cultured in defined and feeder-free conditions, can be differentiated into precursors of mesendoderm and subsequently into cjPGC-like cells (cjPGCLCs) with a transcriptome similar to human PGCs/PGCLCs. Our results not only pave the way for studying PGC development in a non-human primate in vitro under experimentally controlled conditions, but also provide the opportunity to derive functional marmoset gametes in future studies.

A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.

Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.

Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro.

Background: The ribosomal protein S6 kinase 1 (S6K1) is one of the main components of the mTOR/S6K signal transduction pathway, which controls cellular metabolism, autophagy, growth, and proliferation. Overexpression of S6K1 was detected in tumors of different origin including breast cancer, and correlated with the worse disease outcome. In addition, significant accumulation of S6K1 was found in the nuclei of breast carcinoma cells suggesting the implication of kinase nuclear substrates in tumor progression. However, this aspect of S6K1 functioning is still poorly understood. The main aim of the present work was to study the subcellular localization of S6K1 in breast cancer cells with the focus on cell migration. Methods: Multicellular spheroids of MCF-7 cells were generated using agarose-coated Petri dishes. Cell migration was induced by spheroids seeding onto adhesive growth surface and subsequent cultivation for 24 to 72 hours. The subcellular localization of S6K1 was studied in human normal breast and cancer tissue samples, 2D and 3D MCF-7 cell cultures using immunofluorescence analysis and confocal microscopy. Results: Analysis of histological sections of human breast tissue samples revealed predominantly nuclear localization of S6K1 in breast malignant cells and its mainly cytoplasmic localization in conditionally normal cells. In vitro studies of MCF-7 cells demonstrated that the subcellular localization of S6K1 depends on the cell density in the monolayer culture. S6K1 relocalization from the cytoplasm into the nucleus was detected in MCF-7 cells migrating from multicellular spheroids onto growth surface. Immunofluorescence analysis of S6K1 and immunocoprecipitation assay revealed the colocalization and interaction between S6K1 and transcription factor TBR2 (T-box brain protein 2) in MCF-7 cells. Conclusions: Subcellular localization of S6K1 depends on the density and locomotor activity of the MCF-7 cells.