Agency for Science, Technology and Research

National University of Singapore

Agency for Science, Technology and Research (a*star)

Huck Hui Ng

Dominic Jian-Chien Heng

Yock Young Dan

Yun Shen Chan

Yuanyu Hu

Bo Feng

Jianming Jiang

Thomas Lufkin

Petra Kraus

Masafumi Muratani

Hyunsoo Shawn Je

Iwona Szczerbinska

Na Yu Chia

Junghyun Jo

Weiquan John Tng

The present disclosure provides a method of deriving and maintaining a midbrain-like organoid in culture, comprising (a) culturing pluripotent stem cells to obtain neuronal lineage embryoid bodies; (b) culturing the neuronal lineage embryoid bodies from (a) to obtain midbrain regionalized tissues; (c) embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix to obtain neuroepithelial tissues; and (c) culturing the neuroepithelial tissues from (c) to obtain a midbrain-like organoid. Also disclosed herein are culture media suitable for deriving and maintaining neuronal lineage embryoid bodies comprising (a) TGF-β Inhibitor and/or SMAD2/3 inhibitors; and (b) WNT-signaling activator; culture media suitable for deriving and maintaining midbrain regionalized tissues comprising (a) TGF-β Inhibitor and/or SMAD2/3 inhibitors; (b) WNT-signaling activator; (c) hedgehog signaling protein; and (d) fibroblast growth factor; culture media suitable for deriving and maintaining neuroepithelial tissues comprising (a) hedgehog signaling protein; and (b) fibroblast growth factor and culture media suitable for deriving and maintaining a midbrain-like organoid comprising (a) neurotrophin factor; (b) ascorbic acid; and (c) activator of cAMP-dependent pathway.

Generation of midbrain-specific organoids from human pluripotent stem cells

The present invention relates to a cell culture medium for preparing liver, gastric, pancreatic, colon or intestinal adult stem cell isolated from adult tissue, as well as for maintaining such stem cell in the undifferentiated state. The cell culture medium comprises a base medium; an ABL and SRC dual kinase inhibitor/an ABL kinase inhibitor and a SRC kinase inhibitor; a mitogenic factor; a WNT signalling pathway activator; a stimulator for NAD+ and NADP+ generation; and a cAMP/PKA pathway activator. In a particular embodiment, the ABL and SRC dual kinase inhibitor is Dasatinib; the mitogenic factor is EGF; the WNT signalling pathway activator is R-Spondin 1; the stimulator for NAD+ and NADP+ generation is nicotinamide; and the cAMP/PKA pathway activator is cholera endotoxin.

Cell culture medium

This application describes liver stem cells (LSC), and differentiated hepatocytes, cholangiocytes, and 3D cellular structures derived therefrom. Methods for producing LSC and mature, differentiated hepatocytes and cholangiocytes in culture are provided. Also provided are cell culture systems and cell culture media for producing a homogenous population of liver stem cells that remain in an undifferentiated state over multiple passages in culture. The LSC and methods are useful for producing homogenous populations of hepatocytes and cholangiocytes for downstream applications. The LSC can be transplanted into subjects to treat liver diseases.

Derivation of hepatic stem cells and mature liver cell types and uses thereof

A method for culturing and maintaining a pluripotent stem cell in an undifferentiated state is provided. The method comprises culturing the pluripotent stem cell in a medium comprising an MEK inhibitor, a GSK3 inhibitor, a dual inhibitor of AMPK and/or BMP signaling and LIF. A cell produced by the method, cell culture medium and a kit for performing the method described is also provided.

Culturing pluripotent stem cells

According to the invention there is provided methods for inducing pluripotent stem cells in vitro, vectors and compositions for producing the same and methods for using the induced pluripotent stem cell for treating a patient in need of a pluripotent stem cell treatment.

Nuclear receptor and mutant thereof and the use of the same in the reprogramming of cells

A processing method to trim ends of DNA fragments, exposing the internal DNA part to give original DNA sequence information enabling application of next generation sequencing for DNA samples to be amplified by DOP-PCR or other primer dependent amplification methods. Specifically, nucleic acids are amplified using primers comprising a recognition site for a restriction enzyme, for example Bpml or Mmel. Primer sequences are removed by cleavage with the restriction enzyme.

Processing of amplified DNA fragments for sequencing

According to the invention there is provided methods for inducing pluripotent stem cells in vitro, comprising introducing a gene or polypeptide of a nuclear receptor and one or more gene or polypeptide selected from the group consisting of Sox, Kr&#xfc;ppel-like factor or the myc family, to cells in vitro. The present invention also provides vectors and compositions for producing the same and methods for using the induced pluripotent stem cell for treating a patient in need of a pluripotent stem cell treatment.

Methods of inducing pluripotency in human somatic cells and methods of maintaining pluripotency in human embryonic stem cells (hESCs) are provided, as well as cells and uses of employing such cells. The methods comprise culturing cells in the presence of (i) OCT4 and SOX2; (ii) at least one of KLF4 and c-MYC; and at least one of PRDM14 and NFRKB.

Method for inducing pluripotency in human somatic cells with PRDM14 or NFRKB

The invention provides a method of effecting de-differentiation of an at least partially differentiated cell or of maintaining pluripotency and/or self-renewing characteristics of an undifferentiated cell. The method comprises increasing the amount or the activity of an Err protein, or a functional fragment thereof, in the cell.

Growing community of inventors

Singapore, Singapore

Huck Hui Ng