Kawamura, F. | Search bioRxiv

bioRxiv2026

Precursor of chemically expanded hepatocytes (pre-cHep) with 1-million-fold expansion potential and liver repopulation capacity

Primary human hepatocytes (PHHs) are the gold standard for toxicology and drug metabolism studies in industry. However, their limited availability, substantial batch-to-batch variability, and high cost restrict their use. Here, we report a novel culture condition that reprograms PHHs into a proliferative state. These proliferating cells, termed precursors of chemically expanded hepatocytes (pre-cHep), expand over 106-fold within 30 days while retaining liver repopulation capacity comparable to PHHs. pre-cHep can further differentiate into chemically expanded hepatocytes (cHep) as three-dimensional (3D) spheroids within 7 days in vitro, exhibiting global gene expression profiles, albumin production, and cytochrome P450 (CYP) activities similar to 3D-cultured PHH spheroids (3D PHH). Efficient genetic manipulation of pre-cHep using CRISPR/Cas9 is also achievable. Together, pre-cHep and cHep represent a promising alternative to high-quality PHHs, providing a more affordable, reproducible, and scalable source of human hepatocytes for toxicology, drug metabolism studies, disease modelling, towards precision drug development.

bioengineering↗

bioRxiv2018

Magnesium suppresses defects in the formation of 70S ribosomes as well as in sporulation caused by lack of several individual ribosomal proteins.

Individually, the ribosomal proteins L1, L23, L36 and S6 are not essential for cell proliferation of B. subtilis, but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg2+ content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+ content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+ content improved the growth rate of the {Delta}rplA (L1) and the {Delta}rplW (L23) mutant but did not restore those of the {Delta}rpmJ (L36) and the {Delta}rpsF (S6) mutants. The lack of L1 caused a decrease in the production of Spo0A, the master regulator of sporulation, resulting in a decreased sporulation frequency. However, deletion of yhdP and overexpression of mgtE increased the production of Spo0A and partially restored the sporulation frequency in the {Delta}rplA (L1) mutant. These results indicate that Mg2+ can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.\n\nIMPORTANCEWe previously reported that an increase in the cellular Mg2+ content can suppress defects in 70S ribosome formation and growth rate caused by the absence of ribosomal protein L34. In the present study, we demonstrated that even in mutants lacking individual ribosomal proteins other than L34 (L1, L23, L36 and S6), an increase in the cellular Mg2+ content could restore the 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg2+ content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.

genetics↗