Chemical-induced epigenome resetting for regeneration program activation in human cells.

Human somatic cells can be reprogrammed to pluripotent stem cells by small molecules through an intermediate stage with a regeneration signature, but how this regeneration state is induced remains largely unknown. Here, through integrated single-cell analysis of transcriptome, we demonstrate that the pathway of human chemical reprogramming with regeneration state is distinct from that of transcription-factor-mediated reprogramming. Time-course construction of chromatin landscapes unveils hierarchical histone modification remodeling underlying the regeneration program, which involved sequential enhancer recommissioning and mirrored the reversal process of regeneration potential lost in organisms as they mature. In addition, LEF1 is identified as a key upstream regulator for regeneration gene program activation. Furthermore, we reveal that regeneration program activation requires sequential enhancer silencing of somatic and proinflammatory programs. Altogether, chemical reprogramming resets the epigenome through reversal of the loss of natural regeneration, representing a distinct concept for cellular reprogramming and advancing the development of regenerative therapeutic strategies.

Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming.

We recently demonstrated the chemical reprogramming of human somatic cells to pluripotent stem cells (hCiPSCs), which provides a robust approach for cell fate manipulation. However, the utility of this chemical approach is currently hampered by slow kinetics. Here, by screening for small molecule boosters and systematically optimizing the original condition, we have established a robust, chemically defined reprogramming protocol, which greatly shortens the induction time from ∼50 days to a minimum of 16 days and enables highly reproducible and efficient generation of hCiPSCs from all 17 tested donors. We found that this optimized protocol enabled a more direct reprogramming process by promoting cell proliferation and oxidative phosphorylation metabolic activities at the early stage. Our results highlight a distinct chemical reprogramming pathway that leads to a shortcut for the generation of human pluripotent stem cells, which represents a powerful strategy for human cell fate manipulation.

Derivation of totipotent-like stem cells with blastocyst-like structure forming potential.

It is challenging to derive totipotent stem cells in vitro that functionally and molecularly resemble cells from totipotent embryos. Here, we report that a chemical cocktail enables the derivation of totipotent-like stem cells, designated as totipotent potential stem (TPS) cells, from 2-cell mouse embryos and extended pluripotent stem cells, and that these TPS cells can be stably maintained long term in vitro. TPS cells shared features with 2-cell mouse embryos in terms of totipotency markers, transcriptome, chromatin accessibility and DNA methylation patterns. In vivo chimera formation assays show that these cells have embryonic and extraembryonic developmental potentials at the single-cell level. Moreover, TPS cells can be induced into blastocyst-like structures resembling preimplantation mouse blastocysts. Mechanistically, inhibition of HDAC1/2 and DOT1L activity and activation of RARγ signaling are important for inducing and maintaining totipotent features of TPS cells. Our study opens up a new path toward fully capturing totipotent stem cells in vitro.

Chemical reprogramming of human somatic cells to pluripotent stem cells.

Cellular reprogramming can manipulate the identity of cells to generate the desired cell types1-3. The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can enforce somatic cell reprogramming to pluripotent stem cells4-7. By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner8-10. However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome2,11,12 and reduced plasticity13,14; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans.

Chemically defined and xeno-free culture condition for human extended pluripotent stem cells.

Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration.

The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5+ stem cells or Clu+ revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.