Lineage Reprogramming Of Human Gastric Epithelia Into Induced Endodermal Progenitor Cells By Small Molecules And The Underlying Molecular Mechanisms

Stem cells are undifferentiated biological cells that can self-renew and differentiate into specialized functional cells and provide a promising platform for cell therapy, developmental study, drug development and disease moldeling. Embryonic stem cells(ESCs) can self-renew unlimitedly and have the potential to differentiate into all cell types, represent an appealing new source for cell-based therapies. Despite these remarkable advances, some obstacles still remain. Firstly, ESCs is faced with ethical dilemmas and are of immune rejection. Secondly, residual undifferentiated ESCs in the derivatives are tumorigenic. In 2006, Shinya Yamanaka’s lab introduced four pluripotent transcription factors OCT4, SOX2, KLF4, c-MYC and converted fibroblasts into induced pluripotent stem cells(i PSCs). i PSCs circumvent the ethic issues and can be patient-specific that avoid the challenge of immune rejection. However, the residual undifferentiated i PSCs in the derivatives are still tumorigenic. Lineage reprogramming has recently attracted increasing attention as a potential alternative to the directed differentiation of ESCs and i PSCs to obtain cells of a given lineage. Lineage reprogramming has some advantages over i PSCs, for example, nontumorigenicity, pure monolineage cultures of a desired cell type. These features show attractive potential for cell-based therapies. Different means allowing for cell identity switch have been reported. Lineage reprogramming relied initially on the discovery of specific transcription factors(TFs) generally enriched and characteristic of the target cell, and their forced expression in cells of a different fate and bypass pluripotent state. This approach has been successful in various cases, from cells of the hematopoietic systems to neurons, hepatocytes, and cardiomyocytes. However, most of these studies depend on virus-mediate transcription factors overexpresision in starting cells. Those methods are tedious and the integration of virus constructs into resulting cells leads to genomic instability that raises safety concerns. For the purpose of treatment of disease, many researchers are seeking more safe and simple strategies for lineage reprogramming to produce cell types that meet the clinical needs.Small molecules have several prominent advantages over TFs for regulating cell fate: they can be cell permeable, more cost-effective, and more easily synthesized, preserved, and standardized. More importantly, the effects of small molecules can be fine-tuned by varying their concentrations and combinations, thus providing a higher degree of temporal and spatial control over protein function. Small molecules have been widely used in i PSCs reprogramming and lineage reprogramming. Remarkably, Successful reprogramming of mouse fibroblasts to i PSCs by solely using small molecules provided encouraging proof-of-concept evidence. More recently, several groups have demonstrated that small molecules alone are also capable of directly converting fibroblasts or astroglial cells into neurons, neural stem cells and cardiomyocytes, which further demonstrate that chemical reprogramming could be a promising approach to manipulate cell fates. Thus far, there is no report of generation of human i PSCs or any lineage-specific stem/progenitor cells by the use of small molecules alone. To address this issue in endoderm lineage and generate endodermal cell types of clinical value, we select a appropriate cell type-human gastric epithelial cells(h GECs) as starting cells and combine a defined sets of four small molecules(SB431542, Bay K 8644, RG108, BIX01294) with feeder cells- human gastric myofibroblasts(GSEMFs), which serve as reprogramming factors. Under this condition, we successfully converted human h GECs to human induced endodermal progenitor cells(hi Endo PCs) with efficiency of 5%. The hi Endo PCs carry traits of endodermal stem/progenitor cells and clonogenically expand ex vivo. More importantly, hi Endo PCs display the capacity to generate functional endodermal derivatives such as hepatocytes, pancreatic endocrine cells and intestinal epithelial cells. TGFβ inhibition, cell-cycle acceleration and canonical Wnt signaling activation were crucial for this lineage reprogramming. We divided our study into three parts. In the first part, we establish the lineage reprogramming protocol to convert h GECs into hi Endo PCs. In the second part, we characterize the Converted hi Endo PCs in phenotype and differentiation potential. And finally, we explore the mechanism underly the convertion of h GECs to hi Endo PCs.Part Ⅰ Generation of hi Endo PCs from h GECs by Small MoleculesWe isolated h GECs from macroscopically normal mucosa of surgical specimens from patients and cultured in serum-free, low-calcium, hormonally defined Kubota’s medium(KM). We successfully converted h GECs to human induced endodermal progenitor cells(hi Endo PCs) using a mix of defined 8 small molecules(SB43154+VPA +PD0325901+RG108+BIX01294+Bay K 8644+PS48+FBP) in presence of humangastric subepithelial myofibroblasts(GSEMFs) as feeders. We then optimized the 8M cocktail and narrowed to a 4M cocktail with Bay, Bix, RG and SB(BBRS) finally, successful conversions to hi Endo SCs were still obtained with an efficiency of 5%. Moreover, we show that hi Endo PCs are reprogrammed from NCAM+ cells in the presence of the chemicals.Part Ⅱ Characterization of the Converted hi Endo PCsThe hi Endo PCs did not express pluripotency genes(e.g. OCT4, SOX2, NANOG, and C-MYC), nor gastric-associated genes(GAST, MUC6, PGC, and GIF). Transcription factors of endodermal stem/progenitor cells(GATA4, FOXA2, SOX9, HNF1 B, PDX1, ONECUT2, HOXA3) and other stem cell-associated markers(CXCR4, EPCAM, CK19, LGR5) were upregulated in hi Endo PCs compared to h GECs. DNA methylation and Cluster analysis of RNA-seq data revealed that hi Endo PCs were distinct from h GECs. To define the developmental stage of hi Endo PCs, given the natural counterpart to hi Endop Cs cannot be obtained, gene expression in h ESC-derived definitive endoderm(DE), primitive gut-tube endodermal cells(PGT) and posterior foregut(PFG) were used as controls. Results of correlation analysis showed that the relationship of global gene expression of hi Endo PCs to PFG was closer than that to either DE or to PGT in hi Endo PCs. hi Endo PCs can be passaged up to 4-6 times and displayed morphological features of stem cells distinct from those of differentiated h GECs using transmission electron microscopy(TEM). More importantly, the converted hi Endo PCs have the capacities of clonal expansion and of differentiating into multiple derivatives of endodermal cells including functional hepatocytes, pancreatic β cells, intestinal epithelia cells,throid cells and lung cells.Part Ⅲ Signaling Activations Effective for hi Endo PCs ConversionRemarkably, no colony was found when SB431542 was removed, indicating that SB431542 was indispensable for hi Endo PCs reprogramming and maintenance of epithelial phenotype is necessary for the conversion from h GECs to hi Endo PCs. On the other hand, we also find that a sequential EMT-MET exist during hi Endo PCs reprogramming, which correlated with the sequential EMT-MET mechanisms found in early steps of reprogramming from mouse MEFs to i PSCs. The feeder cells(GSEMFs) is also essential for the reprogramming. The paracrine signals secreted from GSEMFs regulated cell cycle, which in turn facilitated hi Endo PCs’ conversion. In addition, Wnt/β-catenin signaling is also activated during reprogramming. Inhibition of Wnt signalling strongly suppressed or abolished reprogramming process. Taken together, the inhibition of EMT by SB431542 or A83-01 maintained the epithelial phenotype of the starting source cells, cell cycle acceleration by secreted factors of GSEMFs, and Wnt signaling activation are essential for the generation of hiEndoPCs.In summary, we have successfully converted human gastric epithelia(h GECs) into induced endodermal progenitor cells(hi Endo PCs) via a small molecule-only lineage reprogramming process for the first time. We validated that hi Endo PCs possess endoderm progenitor properties and revealed the underlying molecular mechanisms in the process of h GECs’ conversion into hi Endo PCs.