Method for expanding stemness and differentiation potential of pluripotent cells. The invention is based on the finding that increasing micro RNA-203 levels in induced pluripotent stem (iPSCs) or embryonic stem (ESCs) cells improves the quality cell fate potential and ability of these cells to differentiate into multiple cell lineages and to reach further maturation properties without interfering with their self-renewal properties. This effect is mediated through the mi R-203-dependent control of de novo DNA methyltransferases Dnmt3a and Dnmt3b, which in turn regulate the methylation landscape of pluripotent cells. The effect can be achieved by overexpression of micro RNA-203 or by adding micro RNA-203 or analogues thereof to the cell culture medium and can be observed using a variety of cellular and in vivo models. The generated cells are naïve pluripotent cells with an improved capacity to differentiate, that can be used to obtain more efficiently differentiated and mature cells proficient for regenerative medicine strategies.

The present invention provides for methods, compositions, and kits for producing an induced pluripotent stem cell from a non-pluripotent mammalian cell using a 3′-phosphoinositide-dependent kinase-1 (PDK1) activator or a compound that promotes glycolytic metabolism as well as other small molecules.

The disclosure provides methods and compositions useful for obtaining induced stem cells, methods of making and use thereof.

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.

The present invention provides an induced pluripotent stem (iPS) cell, or population of iPS cells, wherein the cell or cells giving rise to the iPS cell(s) are obtained from human postpartum tissue or cells, wherein the iPS cell(s) have increased levels of one or more factors selected from Group I: an Oct family member, a Sox family member, a Klf family member, a Myc family member, Nanog, Lin28, and combinations thereof. The present invention also provides differentiated cells derived from the cells of the invention and compositions, including pharmaceutical compositions comprising the cells of the invention. The invention further provides uses of the cells of the invention, e.g., in the treatment of a subject suffering from a disease of disorder. The invention additionally provides methods of generating iPS cell(s) from postpartum tissue, such as the cells of the invention.

Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, Applicants use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, Applicants perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. Applicants further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, Applicants built genetic co-perturbation networks to identify key altered gene modules. Strikingly, we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, highlighting the power of this method to characterize the effects of genetic perturbations. From the fitness responses, Applicants identified ETV2 as a driver of reprogramming towards an endothelial-like state.

The present invention generally provides methods and compositions for transdifferentiation of an animal cell from a first non-pluripotent cell fate to a second non-pluripotent cell fate. Also provided are methods and compositions for the transdifferentiation of an animal cell from a non-pluripotent mesodermal, endodermal, or ectodermal cell fate to a different non-pluripotent mesodermal, endodermal, or ectodermal cell fate.

Provided is a method for producing a stem cell clone, which comprises the steps of: (i) introducing into stem cells an exogenous gene associated with induction of differentiation into somatic cells; (ii) inducing differentiation of the stem cells, introduced with an exogenous gene, into the somatic cells; (iii) dedifferentiating the differentiation-induced somatic cells; and (iv) isolating stem cells having the exogenous gene incorporated into a chromosome thereof from a colony of the stem cells formed in step (iii).

The invention provides cell culture conditions for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs). More particularly, the invention provides a culture platform that allows long-term culture of pluripotent cells in a feeder-free environment; reprogramming of cells in a feeder-free environment; single-cell dissociation of pluripotent cells; cell sorting of pluripotent cells; maintenance of an undifferentiated status; improved efficiency of reprogramming; and generation of a nave pluripotent cell.

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.