The present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell induction factor. The purified RNA preparations are preferably substantially free of RNA contaminant molecules that: i) would activate an immune response in the somatic cells, ii) would decrease expression of the single-stranded mRNA in the somatic cells, and/or iii) active RNA sensors in the somatic cells. In certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded RNAs, un-capped RNA molecules, and/or single-stranded run-on mRNAs.

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 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 current disclosure provides methods for reprogramming mammalian somatic cells by regulating the expression of endogenous cellular genes. Cellular reprogramming of somatic cells can be induced by activating the transcription of embryonic stem cell-associated genes (e.g., oct3/4) and suppressing the transcription of somatic cell-specific and/or cell death-associated genes. The endogenous transcription machinery can be modulated using synthetic transcription factors (activators and suppressors), to allow for faster, and more efficient nuclear reprogramming under conditions amenable for clinical and commercial applications. The current disclosure further provides cells obtained from such methods, along with therapeutic methods for using such cells for the treatment of diseases amendable to stem cell therapy, as well as kits for such uses.

Described herein are methods and compositions related to generation of induced pluripotent stem cells (iPSCs). Improved techniques for establishing highly efficient, reproducible reprogramming using non-integrating episomal plasmid vectors. Using the described reprogramming protocol, one is able to consistently reprogram non-T cells with close to 100% success from non-T cell or non-B cell sources. Further advantages include use of a defined reprogramming media E7 and using defined clinically compatible substrate recombinant human L-521. Generation of iPSCs from these blood cell sources allows for recapitulation of the entire genomic repertoire, preservation of genomic fidelity and enhanced genomic stability.

The present invention describes a virus-derived factor which when provided to cells, e.g., by transfecting the cells with RNA encoding the virus-derived factor, enhances expression of RNA encoding a peptide or protein in the cells. In particular, the virus-derived factor enhances survival of cells, in particular when transfected repetitively with RNA, and reduces an IFN response of cells to transfected RNA. Accordingly, the present invention provides methods and means for enhancing expression of RNA in cells. The cells are preferably transfected with the RNA.

Methods are disclosed herein for efficiently generating human induced pluripotent stem cells (iPSC) containing a nucleic acid including a doxycycline promoter operably linked to a nucleic acid encoding Cas9. These methods include transfecting a human somatic cell with a nucleic acid molecule comprising a doxycycline promoter operably linked to a nucleic acid encoding a Cas9, and constitutive promoter operably linked to a tetracycline responsive element and inducing the somatic cell to form an iPSC, thereby producing an iPSC that can undergo CRISPR/Cas9-mediated recombination at a high efficiency. The human iPSC, or a cell differentiated therefrom, is cultured in the presence of doxycycline to induce expression of the Cas9. These cells can then be used to target in any gene of interest by introducing nucleic acids encoding sgRNAs. Induced pluripotent stem cells produced by these methods are also disclosed.

The present Application is related to methods and compositions for reprogramming adult somatic cells into induced pluripotent stem cells by targeting and remodeling endogenous gene loci without relying on ectopic expression of transcription factors.

Described herein are reprogramming techniques allowing for production of mammary-derived iPSCs (m-iPSCs). The m-iPSCs described herein exhibit all the hallmarks of stem cell identity including round cluster, bright colony morphology, clonal expansion, and pluripotent marker expression (alkaline phosphatase expression, Oct-4, nanog, etc.) Further refined techniques allow for generation of m-iPSCs under essentially defined conditions.

According to the present invention, there are provided a method for producing a human T cell, which comprises the steps of inducing an iPS cell from a human T cell, and differentiating the iPS cell into a T cell; a pharmaceutical composition comprising the T cell produced by the method; and a method for cell-based immunotherapy using the method.