In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.
The generation of human induced pluripotent stem (iPS) cells would represent an appealing option for the derivation of pluripotent patient-specific cells, as no embryos or oocytes are required. However, crucial safety issues have to be addressed in order to create human iPS cells that are clinically useful, as the classical iPS technique involves permanent genetic manipulation that may result in tumor formation. Various experimental strategies have been suggested to accomplish transgene-free derivation of iPS cells, including the use of non-integrating viruses, site specific recombinases to excise transgenes after reprogramming, or RNA transfection. Protein transduction, i.e. the direct delivery of biologically active proteins into cells, has been employed to generate iPS cells but has been found to have very low efficiency. In fact, success of protein transduction is limited by poor stability and solubility of recombinant factors, as well as their poor endosomal release. We recently reported the generation of cell-permeant versions of Oct4 and Sox2 and showed that both can be delivered intracellularly as biologically active proteins. Here we explore conditions for enhanced protein stabilization and delivery into somatic cells. Employing optimized conditions, we demonstrate that Oct4 protein delivery can substitute for Oct4 virus, yielding iPS derivation efficacy comparable to a four virus transduction protocol. The number of colonies is strictly dependent on the dose and duration of cell-permeant Oct4 exposure. We expect our transduction system to reach a thus far unattained level of control over reprogramming activity, turning it into a valuable tool for both the analysis of the reprogramming mechanism and the derivation of transgene-free iPS cells.
Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.
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