Framework nucleic Acid-MicroRNA mediated hepatic differentiation and functional hepatic spheroid development for treating acute liver failure.

The specific induction of hepatic differentiation presents a significant challenge in developing alternative liver cell sources and viable strategies for clinical therapy of acute liver failure (ALF). The past decade has witnessed the blossom of microRNAs in regenerative medicine. Herein, microRNA 122-functionalized tetrahedral framework nucleic acid (FNA-miR-122) has emerged as an unprecedented and potential platform for directing the hepatic differentiation of adipose-derived mesenchymal stem cells (ADMSCs), which offers a straightforward and cost-effective method for generating functional hepatocyte-like cells (FNA-miR-122-iHep). Additionally, we have successfully established a liver organoid synthesis strategy by optimizing the co-culture of FNA-miR-122-iHep with endothelial cells (HUVECs), resulting in functional Hep:HUE-liver spheroids. Transcriptome analysis not only uncovered the potential molecular mechanisms through which miR-122 influences hepatic differentiation in ADMSCs, but also clarified that Hep:HUE-liver spheroids could further facilitate hepatocyte maturation and improved tissue-specific functions, which may provide new hints to be used to develop a hepatic organoid platform. Notably, compared to transplanted ADMSCs and Hep-liver spheroid, respectively, both FNA-miR-122-iHep-based single cell therapy and Hep:HUE-liver spheroid-based therapy showed high efficacy in treating ALF in vivo. Collectively, this research establishes a robust system using microRNA to induce ADMSCs into functional hepatocyte-like cells and to generate hepatic organoids in vitro, promising a highly efficient therapeutic approach for ALF.

Microfluidic 3D hepatic cultures integrated with a droplet-based bioanalysis unit.

A common challenge in microfluidic cell cultures has to do with analysis of cell function without replacing a significant fraction of the culture volume and disturbing local concentration gradients of signals. To address this challenge, we developed a microfluidic cell culture device with an integrated bioanalysis unit to enable on-chip analysis of picoliter volumes of cell-conditioned media. The culture module consisted of an array of 140 microwells with a diameter of 300 m which were made low-binding to promote organization of cells into 3D spheroids. The bioanalysis module contained a droplet generator unit, 15 micromechanical valves and reservoirs loaded with reagents. Each 0.8 nL droplet contained an aliquot of conditioned media mixed with assay reagents. The use of microvalves allowed us to load enzymatic assay and immunoassay into sequentially generated droplets for detection of glucose and albumin, respectively. As a biological application of the microfluidic device, we evaluated hormonal stimulation and glucose consumption of hepatic spheroids. To mimic physiological processes occurring during feeding and fasting, hepatic spheroids were exposed to pancreatic hormones, insulin or glucagon. The droplet-based bioanalysis module was used to measure uptake or release of glucose upon hormonal stimulation. In the future, we intend to use this microfluidic device to mimic and measure pathophysiological processes associated with hepatic insulin resistance and diabetes in the context of metabolic syndrome.

PDGF Receptors and Signaling Are Required for 3D-Structure Formation and Differentiation of Human iPSC-Derived Hepatic Spheroids.

Human iPSC-derived liver organoids (LO) or hepatic spheroids (HS) have attracted widespread interest, and the numerous studies on them have recently provided various production protocols. However, the mechanism by which the 3D structures of LO and HS are formed from the 2D-cultured cells and the mechanism of the LO and HS maturation remain largely unknown. In this study, we demonstrate that PDGFRA is specifically induced in the cells that are suitable for HS formation and that PDGF receptors and signaling are required for HS formation and maturation. Additionally, in vivo, we show that the localization of PDGFRα is in complete agreement with mouse E9.5 hepatoblasts, which begin to form the 3D-structural liver bud from the single layer. Our results present that PDGFRA play important roles for 3D structure formation and maturation of hepatocytes in vitro and in vivo and provide a clue to elucidate the hepatocyte differentiation mechanism.

Bioinspired Sandcastle Worm-Derived Peptide-Based Hybrid Hydrogel for Promoting the Formation of Liver Spheroids.

The generation of hepatic spheroids is beneficial for a variety of potential applications, including drug development, disease modeling, transplantation, and regenerative medicine. Natural hydrogels are obtained from tissues and have been widely used to promote the growth, differentiation, and retention of specific functionalities of hepatocytes. However, relying on natural hydrogels for the generation of hepatic spheroids, which have batch to batch variations, may in turn limit the previously mentioned potential applications. For this reason, we researched a way to establish a three-dimensional (3D) culture system that more closely mimics the interaction between hepatocytes and their surrounding microenvironments, thereby potentially offering a more promising and suitable system for drug development, disease modeling, transplantation, and regenerative medicine. Here, we developed self-assembling and bioactive hybrid hydrogels to support the generation and growth of hepatic spheroids. Our hybrid hydrogels (PC4/Cultrex) inspired by the sandcastle worm, an Arg-Gly-Asp (RGD) cell adhesion sequence, and bioactive molecules derived from Cultrex BME (Basement Membrane Extract). By performing optimizations to the design, the PC4/Cultrex hybrid hydrogels can enhance HepG2 cells to form spheroids and express their molecular signatures (e.g., Cyp3A4, Cyp7a1, A1at, Afp, Ck7, Ck1, and E-cad). Our study demonstrated that this hybrid hydrogel system offers potential advantages for hepatocytes in proliferating, differentiating, and self-organizing to form hepatic spheroids in a more controllable and reproducible manner. In addition, it is a versatile and cost-effective method for 3D tissue cultures in mass quantities. Importantly, we demonstrate that it is feasible to adapt a bioinspired approach to design biomaterials for 3D culture systems, which accelerates the design of novel peptide structures and broadens our research choices on peptide-based hydrogels.