Carbon Fibers as a New Type of Scaffold for Midbrain Organoid Development.

The combination of induced pluripotent stem cell (iPSC) technology and 3D cell culture creates a unique possibility for the generation of organoids that mimic human organs in in vitro cultures. The use of iPS cells in organoid cultures enables the differentiation of cells into dopaminergic neurons, also found in the human midbrain. However, long-lasting organoid cultures often cause necrosis within organoids. In this work, we present carbon fibers (CFs) for medical use as a new type of scaffold for organoid culture, comparing them to a previously tested copolymer poly-(lactic-co-glycolic acid) (PLGA) scaffold. We verified the physicochemical properties of CF scaffolds compared to PLGA in improving the efficiency of iPSC differentiation within organoids. The physicochemical properties of carbon scaffolds such as porosity, microstructure, or stability in the cellular environment make them a convenient material for creating in vitro organoid models. Through screening several genes expressed during the differentiation of organoids at crucial brain stages of development, we found that there is a correlation between PITX3, one of the key regulators of terminal differentiation, and the survival of midbrain dopaminergic (mDA) neurons and tyrosine hydroxylase (TH) gene expression. This makes organoids formed on carbon scaffolds an improved model containing mDA neurons convenient for studying midbrain-associated neurodegenerative diseases such as Parkinson's disease.

Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons.

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson's Disease.

Organoids are becoming particularly popular in modeling diseases that are difficult to reproduce in animals, due to anatomical differences in the structure of a given organ. Thus, they are a bridge between the in vitro and in vivo models. Human midbrain is one of the structures that is currently being intensively reproduced in organoids for modeling Parkinson's disease (PD). Thanks to three-dimensional (3D) architecture and the use of induced pluripotent stem cells (iPSCs) differentiation into organoids, it has been possible to recapitulate a complicated network of dopaminergic neurons. In this work, we present the first organoid model for an idiopathic form of PD. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and patients with the idiopathic form of PD by transduction with Sendai viral vector. iPSCs were differentiated into a large multicellular organoid-like structure. The mature organoids displayed expression of neuronal early and late markers. Interestingly, we observed statistical differences in the expression levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patient and healthy volunteer. The obtained results show immense potential for the application of 3D human organoids in studying the neurodegenerative disease and modeling cellular interactions within the human brain.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

TRIM28 and Interacting KRAB-ZNFs Control Self-Renewal of Human Pluripotent Stem Cells through Epigenetic Repression of Pro-differentiation Genes.

Reprogramming to induced pluripotent stem cells (iPSCs) and differentiation of pluripotent stem cells (PSCs) are regulated by epigenetic machinery. Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate both processes; however, the exact mechanism and identity of participating KRAB-ZNF genes remain unknown. Here, using a reporter system, we show that TRIM28/KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to cause stable gene repression during reprogramming. Using several expression datasets, we identified KRAB-ZNFs (ZNF114, ZNF483, ZNF589) in the human genome that maintain pluripotency. Moreover, we identified target genes repressed by these KRAB-ZNFs. Mechanistically, we demonstrated that these KRAB-ZNFs directly alter gene expression of important developmental genes by modulating H3K9me3 and DNA methylation of their promoters. In summary, TRIM28 employs KRAB-ZNFs to evoke epigenetic silencing of its target differentiation genes via H3K9me3 and DNA methylation.