Disclosed herein are methods of inducing and/or promoting cardiomyocyte maturation comprising: providing an immature cardiomyocyte; providing a three dimensional (3D) cardiac extracellular matrix (ECM) scaffold; and inducing and/or promoting cardiomyocyte cell maturation by seeding the immature cardiomyocyte in the 3D cardiac ECM scaffold and harvesting once the cardiomyocyte has reached maturity. Also disclosed herein are methods of treating a disease in a mammal comprising transplanting a mature cardiomyocyte into an ischemic heart, wherein the mature cardiomyocyte is generated comprising the steps of: providing an immature cardiomyocyte; providing a 3D cardiac ECM scaffold; and generating mature cardiomyocyte by seeding the immature cardiomyocyte in a 3D cardiac ECM scaffold or co-culturing the immature cardiomyocyte in the presence of endothelial cells or stromal cells; and harvesting once the cardiomyocyte has reached maturity.

A method for obtaining a plurality of pluripotent adult olfactory stem cells (APOSCs) includes isolating the APOSCs, culturing the isolated APOSCs in a sphere culture medium, and collecting the cultured APOSCs that express Bmi-1 (B-lymphoma moloney murine leukemia virus insertion region-1), Oct-4 (Octamer-binding transcription factor 4), Sox-2 (Sex-determining region Y (SRY)-box 2), Nanog, SSEA-4 (Stage-specific embryonic antigen-4), ki67, c-Myc, KLF-4 (Kruppel Like Factor 4), K14 (Cytokeratin 14) and ICAM-1 (Intercellular Adhesion Molecule 1).

Preparation and expansion methods for human pluripotent stem cell-derived human retinal pigment epithelial cells are provided. The preparation method includes the following steps: collecting 3D-PRE spheroids derived from human pluripotent stem cells, performing mechanical separation to remove non-RPE cells and clusters which are non-pigmented and retain a pigmented RPE cell sheet, enzymatically digesting the pigmented RPE cell sheet to obtain an RPE single cell suspension, and thereby the human pluripotent stem cell-derived human retinal pigment epithelial cells are obtained. The expansion method includes centrifuging the RPE single cell suspension and removing a supernatant, resuspending in an RPE cell medium and seeding into a cell culture container precoated with extracellular matrix to allow primary culture, and subculturing the cells after cells spread out.

The generation of complex organ tissues from human embryonic and pluripotent stem cells (PSCs) remains a major challenge for translational studies. It is shown that PSCs can be directed to differentiate into intestinal tissue in vitro by modulating the combinatorial activities of several signaling pathways in a step-wise fashion, effectively recapitulating in vivo fetal intestinal development. The resulting intestinal organoids were three-dimensional structures consisting of a polarized, columnar epithelium surrounded by mesenchyme that included a smooth muscle-like layer. The epithelium was patterned into crypt-like SOX9-positive proliferative zones and villus-like structures with all of the major functional cell types of the intestine. The culture system is used to demonstrate that expression of NEUROG3, a pro-endocrine transcription factor mutated in enteric anendocrinosis is sufficient to promote differentiation towards the enteroendocrine cell lineage. In conclusion, PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development, homeostasis and disease.

Treatment of rheumatoid arthritis has been revolutionized by the introduction of TNF-alpha blockers, which represent a 40 billion dollar annual market. Unfortunately, a significant proportion of patients fail to respond to these treatments. The current invention provides the use of stem cell conditioned T cells to overcome resistance to TNF-alpha blockers by concurrently providing a source of immune modulatory and regenerative cells that synergize with TNF-alpha blockers.

A method of producing helper T cells, comprising: (i) culturing T cells, which have been induced from pluripotent stem cells and into which a CD4 gene or a gene product thereof has been introduced, in a medium containing IL-2 and IL-15; and (ii) isolating CD40L-highly expressing T cells from cells obtained in step (i).

In the context of a stem cell-based therapy in which differentiated cells are derived from pluripotent stem cells, this application discloses approaches for eliminating residual pluripotent stem cells that may remain in a batch of differentiated progeny cells. This advantageously prevents the formation of teratoma tumors when the differentiated cells are eventually used for the therapy. This can be accomplished by exposing the batch of differentiated progeny cells and the residual pluripotent stem cells to an alternating electric field for a period of time. The frequency and field strength of the alternating electric field are such that the pluripotent stem cells die off as a result of exposure to the alternating electric fields, while the differentiated cells remain substantially unharmed. This results in a purified batch of differentiated progeny cells that is rendered safe for use in the stem cell-based therapy.

Provided herein are methods and systems for bio-printing of three-dimensional organs and organoids. Also provided herein are bio-printed three-dimensional organs and organoids for use in the generation and/or the assessment of immunological products and/or immune responses. Also provided herein are methods and system for bio-printing three-dimensional matrices.

In related-art methods of differentiating pluripotent stem cells into a desired cell type, there has not been established a differentiation induction method using human ES/iPS cells and being stable and highly efficient. A method of inducing differentiation into a desired cell type within a short period of time and with high efficiency by attenuating differentiation resistance of a pluripotent stem cell to generate a pluripotent stem cell that actively proceeds to a differentiated cell type has been found, and thus the present invention has been completed.

Provided herein are methods and systems for bio-printing of three-dimensional organs and organoids. Also provided herein are bio-printed three-dimensional organs and organoids for use in the generation and/or the assessment of immunological products and/or immune responses. Also provided herein are methods and system for bio-printing three-dimensional matrices.