Generation of induced pluripotent stem cell line RCPCMi008-A derived from patient with spinocerebellar ataxia 17.

IPSC line RCPCMi004-8 was generated from skin fibroblasts collected from a male patient with spinocerebellar ataxia 17. The patient has expanded trinucleotide CAG repeats in the TBP (TATA-binding protein) gene on chromosome 6q27. The reprogramming of fibroblasts was performed with Sendai viruses containing Oct-4, Sox-2, Klf-4, and c-Myc. Pluripotency was confirmed by immunofluorescence, RT-PCR, and the formation of embryoid bodies. The RCPCMi008-A cell line carries the same trinucleotide CAG repeats in the TBP gene. The RCPCMi008-A cell line can be used to model Spinocerebellar ataxia in vitro.

"Necessity Is the Mother of Invention" or Inexpensive, Reliable, and Reproducible Protocol for Generating Organoids.

Organoids are three-dimensional (3D) cell cultures that replicate some of the key features of morphology, spatial architecture, and functions of a particular organ. Organoids can be generated from both adult and pluripotent stem cells (PSCs), and complex organoids can also be obtained by combining different types of cells, including differentiated cells. The ability of pluripotent cells to self-organize into organotypic structures containing several cell subtypes specific for a particular organ was used for creating organoids of the brain, eye, kidney, intestine, and other organs. Despite the advantages of using PSCs for obtaining organoids, an essential shortcoming that prevents their widespread use has been a low yield when they are obtained from a PSC monolayer culture and a large variation in size. This leads to great heterogeneity on further differentiation. In this article, we describe our own protocol for generating standardized organoids, with emphasis on a method for generating brain organoids, which allows scaling-up experiments and makes their cultivation less expensive and easier.

Differentiation of Human Pluripotent Stem Cells into Mesodermal and Ectodermal Derivatives Is Independent of the Type of Isogenic Reprogrammed Somatic Cells.

Induced pluripotent stem cells (iPSCs) have the capacity to unlimitedly proliferate and differentiate into all types of somatic cells. This capacity makes them a valuable source of cells for research and clinical use. However, the type of cells to be reprogrammed, the selection of clones, and the various genetic manipulations during reprogramming may have an impact both on the properties of iPSCs and their differentiated derivatives. To assess this influence, we used isogenic lines of iPSCs obtained by reprogramming of three types of somatic cells differentiated from human embryonic stem cells. We showed that technical manipulations in vitro, such as cell sorting and selection of clones, did not lead to the bottleneck effect, and that isogenic iPSCs derived from different types of somatic cells did not differ in their ability to differentiate into the hematopoietic and neural directions. Thus, the type of somatic cells used for the generation of fully reprogrammed iPSCs is not important for the practical and scientific application of iPSCs.

[Genetic Cell Reprogramming: A New Technology for Basic Research and Applied Usage].

Gene function disclosure and the development of modern technologies of genetic manipulations offered the possibility of genetic reprogramming application to alter cell specialization. With the involvement of a gene set that encodes the transcription factors responsible for the pluripotent state, any cell of an adult body could be reprogrammed into the embryonal.state and pluripotency could be induced in this cell. Such reprogrammed cells were called induced pluripotent stem cells (iPSCs), and they are capable of again passing through all developmental stages. This provides new possibilities for studies of the basic mechanisms of developmental biology, the formation of specific cell types, and the whole body. In culture, iPSCs could be maintained permanently in a nontransformed state and permit genetic manipulations while maintaining their pluripotent properties. Such a unique combination of their properties makes them an attractive tool for studies of various pathologies and for the delineation of treatment approaches. This review discusses the basic and applied aspects of iPSCs biology.

Molecular barriers to processes of genetic reprogramming and cell transformation.

Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar "molecular barriers" that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation.