Enhanced Differentiation of Adult Bone Marrow-Derived Stem Cells to Liver Lineage in Aggregate Culture

Abstract

Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology.

Enhanced hepatic differentiation of rat bone marrow-derived mesenchymal stem cells in spheroidal aggregate culture on a decellularized liver scaffold

“…In the present study, a natural 3D scaffold from decellularized liver matrix was used to optimize the differentiation of MSC spheroids to functional hepatocytes. Our data demonstrated that the 3D biomatrix scaffold as well as spheroid culture promotes the induction of lineage-specific differentiation of MSCs into hepatocyte-like cells, which is consistent with the findings of other studies ( 15 – 17 , 20 , 21 ). Moreover, we suggest that the combination of cell-cell interactions and ECM plays a positive role in the differentiation of MSCs to hepatocyte-like cells.…”

“…To prevent necrosis of the core caused by a deficiency of oxygen and nutrients, the diameter of the spheroid was controlled by the cell seeding density, culture time and the rocking speed. Previous 3D spheroid culture systems often involved a cell-accumulation technique in microwells with certain types of cells ( 15 , 16 ). However, these culture systems experienced difficulties in terms of producing sufficient numbers of MSC spheroids.…”

“…Three-dimensional (3D) culture systems have been shown to promote enhanced cellular structure and function in many types of cells and tissues, including mammary epithelial cells, MSCs as well as neural and hepatic cells ( 12 – 14 ). MSCs cultured as 3D spheroids in suspension are characterized by enhanced levels of differentiation and higher degrees of maturity of MSC-derived hepatocytes compared with traditional adherent monolayer cultures ( 15 – 17 ).…”

Differentiation and Selection of Hepatocyte Precursors in Suspension Spheroid Culture of Transgenic Murine Embryonic Stem Cells

“…In line with this, enhanced differentiation of adult bone marrow-derived stem cells to liver lineage in a three-dimensional aggregate culture in comparison to adherent monolayer cultures has been demonstrated [88]. Taking into account that no special medium or substances facilitating hepatic differentiation were applied in our experimental set, we assume that a similar mechanism significantly contributed to the liver fetal cell-like differentiation of spheroid cells.…”

Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon

“…It is also suspected that the reprogrammed cells that underwent gene removal and FCM processes were susceptible to a decrease of viability and acquired hepatocyte function, whereas the cell-cell interactions in the spheroidal iHeps supported the maintenance of viability and promotion of differentiation into hepatocytes 39 40 41 . Thus, 3D culture environments including spheroids are expected to contribute to the maintenance and maturation of iHeps as reported for MSCs, iPSCs, and ESCs 42 43 44 , which could be an interesting avenue for future research.…”