The present invention provides a method for inducing an inversion of normal blood coagulation factor VIII (F8) gene, a method for correcting an inversion of blood coagulation factor VIII gene in which the inversion has occurred, and a Hemophilia A patient-derived induced pluripotent stem cell in which the inversion is corrected, constructed using the same. The method of the present invention effectively reproduces the inversion of intron 1 and intron 22 of the F8 gene, which is responsible for the majority of severe hemophilia A, and thereby may be effectively used for studying the development mechanism of hemophilia A and as a research tool for screening therapeutic agents. The inversion-corrected induced pluripotent stem cell constructed according the method of the present invention enables an efficient and fundamental treatment for hemophilia A by restoring a genotype in which mutation has occurred to a wild type-like state, without limitation via normal gene or protein delivery.

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

The nuclear reprogramming of somatic cells with mRNA encoding reprogramming factors is shown to be greatly accelerated by activation of innate immune responses in the somatic cell. Methods of activating innate immunity include activation of PKR, of toll-like receptors, e.g. TLR3, etc. In some embodiments the mRNA provides the activator of innate immunity.

The present invention provides methods for improving the efficiency of inducing pluripotent stem cells, as well as vectors and compositions for use therein. In the induction of pluripotent stem cells which contains the step of introducing a vector that contains the KLF gene, OCT gene, and SOX gene in this order, the efficiency of pluripotent stem cell induction was successfully increased significantly by further introducing a vector that contains the KLF gene but not the OCT gene and the SOX gene. The methods of the present invention have an excellent feature in that they allow efficient induction of pluripotent stem cells under a temperature condition closer to the physiological environment, and prompt vector removal after the pluripotent stem cell induction. The present invention enables more efficient induction of pluripotent stem cells.

Provided herein are methods for the efficient in vitro differentiation of somatic cell-derived pluripotent stem cells to hematopoietic precursor cells, and the further differentiation of the hematopoietic precursor cells into immune cells of various myeloid or lymphoid lineages, particularly T cells, NK cells, and dendritic cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the hematopoietic precursor cells.

Provided herein are methods for the efficient in vitro differentiation of somatic cell-derived pluripotent stem cells to hematopoietic precursor cells, and the further differentiation of the hematopoietic precursor cells into immune cells of various myeloid or lymphoid lineages, particularly T cells, NK cells, and dendritic cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the hematopoietic precursor cells.

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

This invention provides a method for preparing a brown adipocyte from a somatic cell of a mammal by introducing at least one brown adipocyte-related gene or expression product thereof and at least one reprogramming-related gene or expression product thereof into the somatic cell, the brown adipocyte-related gene being at least one member selected from the group consisting of PRDM16(P) and C/EBP(C), the reprogramming-related gene being at least one member selected from the group consisting of Myc family genes (c-Myc(M), N-Myc, L-Myc(L), S-Myc, and B-Myc), GLIS family genes (GLIS1 (G), GLIS 2, and GLIS 3), Klf family genes (KLF1, KLF2, KLF3, KLF4(K), KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, KLF16, and KLF17), Oct family genes, Sox family genes, and Lin-28.

The present disclosure generally regards methods and compositions for providing multi-lineage hematopoietic precursor cells from pluripotent stem cells (PSCs). The PSCs comprise an expression construct encoding an ETS/ERG gene, GATA2 and HOXA9. Also provided are methods for providing hematopoietic stem cells capable of long-term engraftment in mammals, such as humans. Further provided are therapeutic compositions including the provided hematopoietic stem cells and precursors of hematopoietic cells, and methods of using such for the treatment of subjects.

The present invention includes a method of preparing differentiated endodermal cells comprising growing human pluripotent stem cells (hPSC) under serum free conditions for 3 days on a layer of fibronectin or a gelatinous protein layer in the presence of high activin/TGF- with a GSK3 inhibitor and a PI3K/mTORC inhibitor in a basal differentiation medium; adding a knock out serum replacement to the media starting on day 4 through day 7; and harvesting the endodermal cells grown therein.