Abstract: The present disclosure provides methods and reagents for culturing pluripotent stem cells. The method comprises a first step of culturing in a medium comprising inhibitors of GSK-3?, JNK, p38, PKC and Erk1/2, followed by a second step of culturing in a medium comprising inhibitors of GSK-3?, JNK, p38 and PKC, and not comprising an inhibitor of Erk1/2. The medium of the second step may also further comprise dextran sulfate. The pluripotent stem cells produced using the methods and reagents provided may also be differentiated into a cell type of interest. The methods and reagents provided in this disclosure offer robust and scalable technologies for manufacturing the quantities of cells anticipated to be required for widespread patient access.

Abstract: The present disclosure provides a method for generating progenitor T cells from stem and/or progenitor cells comprising exposing the stem and/or progenitor cells to Notch ligand Delta-like-4 (DL4) and vascular adhesion molecule 1 (VCAM-1) under conditions suitable to generate progenitor T cells. The method provided is suitable for in vitro and in vivo pro-T cell generation. In vitro, the pro-T cells are generated under serum-free conditions. Cells produced using the method are provided as well as methods of using same.

Abstract: The present inventors identified aggregation of embryonic stem cells and embryoid bodies (EBs) as the cause of the difficulty in generating large numbers of the embryonic stem cells (ES) cell-derived tissues. To counter this, the invention provides a novel bioprocess where aggregation of spheroid forming cells, such as embryonic stem cells and spheroids, such as EBs is controlled, such as by encapsulation of within a matrix. As a result, EBs can be generated with high efficiency and cultured in high cell density, well-mixed systems. Well-mixed conditions facilitate measurement and control of the bulk media conditions and allow for the use of scalable bioreactor systems for clinical production of tissue. Therefore, the invention enables generation of ES cell-derived tissue on a clinical scale. The invention is also applicable to any spheroid-forming cells and other types of pluripotent cells.

Abstract: The present inventors identified aggregation of embryonic stem cells and embryoid bodies (EBs) as the cause of the difficulty in generating large numbers of the embryonic stem cells (ES) cell-derived tissues. To counter this, the invention provides a novel bioprocess where aggregation of spheroid forming cells, such as embryonic stem cells and spheroids, such as EBs is controlled, such as by encapsulation of within a matrix. As a result, EBs can be generated with high efficiency and cultured in high cell density, well-mixed systems. Well-mixed conditions facilitate measurement and control of the bulk media conditions and allow for the use of scalable bioreactor systems for clinical production of tissue. Therefore, the invention enables generation of ES cell-derived tissue on a clinical scale. The invention is also applicable to any spheroid-forming cells and other types of pluripotent cells.