Human Hepatocytes with Drug Metabolic Function Induced from Fibroblasts by Lineage Reprogramming

SummaryObtaining fully functional cell types is a major challenge for drug discovery and regenerative medicine. Currently, a fundamental solution to this key problem is still lacking. Here, we show that functional human induced hepatocytes (hiHeps) can be generated from fibroblasts by overexpressing the hepatic fate conversion factors HNF1A, HNF4A, and HNF6 along with the maturation factors ATF5, PROX1, and CEBPA. hiHeps express a spectrum of phase I and II drug-metabolizing enzymes and phase III drug transporters. Importantly, the metabolic activities of CYP3A4, CYP1A2, CYP2B6, CYP2C9, and CYP2C19 are comparable between hiHeps and freshly isolated primary human hepatocytes. Transplanted hiHeps repopulate up to 30% of the livers of Tet-uPA/Rag2−/−/γc−/− mice and secrete more than 300 μg/ml human ALBUMIN in vivo. Our data demonstrate that human hepatocytes with drug metabolic function can be generated by lineage reprogramming, thus providing a cell resource for pharmaceutical applications

A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts

Terminally differentiated cells can be generated by lineage reprogramming, which is, however, hindered by incomplete conversion with residual initial cell identity and partial functionality. Here, we demonstrate a new reprogramming strategy by mimicking the natural regeneration route, which permits generating expandable hepatic progenitor cells and functionally competent human hepatocytes. Fibroblasts were first induced into human hepatic progenitor-like cells (hHPLCs), which could robustly expand in vitro and efficiently engraft in vivo. Moreover, hHPLCs could be efficiently induced into mature human hepatocytes (hiHeps) in vitro, whose molecular identity highly resembles primary human hepatocytes (PHHs). Most importantly, hiHeps could be generated in large quantity and were functionally competent to replace PHHs for drug-metabolism estimation, toxicity prediction and hepatitis B virus infection modeling. Our results highlight the advantages of the progenitor stage for successful lineage reprogramming. This strategy is promising for generating other mature human cell types by lineage reprogramming.</p

Long-term functional maintenance of primary human hepatocytes in vitro

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.</p

Guideline for the extraction, isolation, purification, and structural characterization of polysaccharides from natural resources

Abstract Polysaccharide is widely distributed in natural resources, and currently attracts scientists' attention for their various bio‐functions including immunomodulatory activity, hypoglycemic activity, hypolipidemic activity, antitumor activity, promoting effects on gastrointestinal function, and so on. The purity and structure characteristics of different polysaccharides greatly restrict the in‐depth study of their bioactivity mechanism. Therefore, this guideline will display the extraction, isolation, purification, and structural characterization of polysaccharides from natural resources. The extraction methods of acid‐soluble polysaccharides, water‐soluble polysaccharides, and alkaline soluble polysaccharides are included. Common purification methods like membrane filtration, alcohol precipitation, and column chromatographic separation are explained in detail. A comprehensive procedure for polysaccharide structural characterization is given from molecular weight and monosaccharide composition study to Fourier transform infrared and nuclear magnetic resonance analysis. Examples are added where necessary to help you better understand this guideline

Nephropathy is aggravated by fatty acids in diabetic kidney disease through tubular epithelial cell necroptosis and is alleviated by an RIPK-1 inhibitor

Introduction: Diabetic kidney disease (DKD), one of the leading causes of end-stage renal disease, has complex pathogenic mechanisms and few effective clinical therapies. DKD progression is accompanied by the loss of renal resident cells, followed by chronic inflammation and extracellular matrix deposition. Necroptosis is a newly discovered form of regulated cell death and is a major form of intrinsic cell loss in certain diabetic complications such as cardiomyopathy, intestinal disease, and retinal neuropathy; however, its significance in DKD is largely unknown. Methods: In this study, the expression of necroptosis marker phosphorylated MLKL (p-MLKL) in renal biopsy tissues of patients with DKD was detected using immunofluorescence and semiquantified using immunohistochemistry. The effects of different disease-causing factors on necroptosis activation in human HK-2 cells were evaluated using immunofluorescence and western blotting. db/db diabetic mice were fed a high-fat diet to establish an animal model of DKD with significant renal tubule damage. Mice were treated with RIPK1 inhibitor RIPA-56 to evaluate its renal protective effects. mRNA transcriptome sequencing was used to explore the changes in signaling pathways after RIPA-56 treatment. Oil red O staining and electron macroscopy were used to observe lipid droplet accumulation in renal biopsy tissues and mouse kidney tissues. Results: Immunostaining of phosphorylated RIPK1/RIPK3/MLKL verified the occurrence of necroptosis in renal tubular epithelial cells (RTECs) of patients with DKD. The level of the necroptosis marker p-MLKL correlated positively with the severity of renal functional, pathological damages, and lipid droplet accumulation in patients with DKD. High glucose and fatty acids were the main factors causing necroptosis in human renal tubular HK-2 cells. Renal function deterioration and renal pathological injury were accelerated, and the necroptosis pathway was activated in db/db mice fed a high-fat diet. Application of RIPA-56 effectively reduced the degree of renal injury, inhibited the necroptosis pathway activation, and reduced necroinflammation and lipid droplet accumulation in the renal tissues of db/db mice fed a high-fat diet. Conclusion: The present study revealed the role of necroptosis in the progression of DKD and might provide a new therapeutic target for the treatment of DKD