Effective three‐dimensional culture of hepatocyte‐like cells generated from human adipose‐derived mesenchymal stem cells

Abstract

Background:The aim of this study was to clarify the effectiveness of a new threedimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs). Methods: Human ADSCs (2 × 10 4 ) with or without 0.1 mg/mL human recombinant peptide μ-piece per well were seeded in a 96-well U-bottom plate and then our threestep differentiation protocol was applied for 21 days. At each step, cell morphology and gene expression were investigated. Mature hepatocyte functions were evaluated after HLC differentiation. These parameters were compared between 2D-and 3Dcultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl 4 induced acute liver failure model mice. Results: Two-dimensional-cultured HLCs at day 21 did not have a spindle shape and had formed spheroids after day 6, which gradually increased in size for 3D-cultured HLCs. Definitive endoderm, hepatoblast, and hepatocyte genes showed significantly higher expression in the 3D culture group. Three-dimensional-cultured HLCs also had higher albumin expression, CYP3A4 activity, urea synthesis, and ammonium metabolism, and much higher expression of ion transporter, blood coagulation, and cell communication genes. HLC transplantation improved serum liver function, especially in T-Bil levels, and engrafted into immunodeficient mice with HLA class I positive staining. Conclusion:Our new 3D culture protocol is effective to improve hepatocyte functions. Our HLCs might be promising for clinical cell transplantation to treat metabolic disease.

Clinical Application of Induced Hepatocyte-like Cells Produced from Mesenchymal Stromal Cells: A Literature Review

“…Ammonia was reduced by approximately 35% in one study, while BUN was reduced by an average of 35%. Total bilirubin was reduced by approximately 35% in the two studies in which it was reported [43,44].…”

“…Therapeutic utility of mHeps has been demonstrated in a variety of different types of liver disease (Table 2). Several studies have demonstrated the engraftment of these mHeps in rodent models [19,27,28,36,[39][40][41][42][43][44][45] and pigs [46]. The efficiency of engraftment has generally not been reported, although Aurich et al presented data indicating that upwards of 20% of mouse livers were composed of mHeps at 10 weeks [41].…”

“…The modeling of the therapeutic applications of these cells is summarized in Table 2. In these studies, the carbon tetrachloride (CCl 4 ) model of liver injury using a single dose to induce an acute injury in immunocompromised mice is the most often-used model [22,[25][26][27][28][43][44][45]. Other models employed TK-NOG mice (immunocompromised mice with the thymidine kinase gene expressed via the albumin promoter [49]) treated with ganciclovir [19], partial hepatectomy [38][39][40]45], galactosamine/lipopolysaccharide [35], galactosamine [50] and acetaminophen toxicity [36].…”

“…Seven of the 16 studies identified with clinically relevant results used the CCI 4 model and reported the engraftment of cells, either in the liver or under the kidney capsule [22,26,36,[38][39][40]42]. Of these, six of seven studies reported an improvement in clinical parameters, including reduction in total bilirubin, reductions in serum ammonia or blood urea nitrogen (BUN), and reduced aspartate aminotransferase (AST)/alanine amino transferase (ALT) levels [22,[26][27][28]43,44]. AST/ALT levels were reduced by approximately 40% compared to untreated controls in these experiments.…”

Newly Generated 3D Schwann-Like Cell Spheroids From Human Adipose-Derived Stem Cells Using a Modified Protocol

“…The RCP μ-piece acts as a scaffold for ADSCs and can support the formation of a large cell cluster. The features of this RCP include xeno-antigen-free characteristics, high cell adhesiveness, biodegradation absorption, clinical-grade quality, and stable manufacturing quality 23 . Therefore, it can achieve a cell aggregate-like technology.…”