Therapeutic potential of bone-marrow-derived mesenchymal stem cells differentiated with growth-factor-free coculture method in liver-injured rats.

Mesenchymal stem cell (MSC) differentiation by growth factors may be improper due to possibility of clinical risk. We have previously developed a growth-factor-free coculture method and observed rat MSCs differentiated into hepatic progenitor cells. This study was aimed to validate hepatic differentiation potential in vivo. MSCs from bone marrow of green fluorescent protein-transgenic Sprague-Dawley rats were cocultured with hepatocytes from normal Sprague-Dawley rats, sharing growth-factor-free media. After 14 days, cells were implanted into the spleen of carbon tetrachloride (CCl4)-injured rats and kept for 4 weeks. Fibrosis remarkably decreased in CCl4/cocultured MSC at weeks 1, 3, and 4. Immunohistochemistry revealed that albumin, alpha-fetoprotein, and cytokeratin 19 (CK19) expression was high in CCl4/cocultured MSC only at week 1. Reverse transcription-polymerase chain reaction and Western blot revealed that CCl4/cocultured MSC had reduced alpha-fetoprotein expression at week 4, whereas CK18 and CK19 exhibited stronger expression. Albumin in CCl4/cocultured MSC increased at week 4 only in protein level. We assume that cocultured MSCs had stayed at hepatic progenitor stage until week 3, and differentiated into hepatocytes or bile-ductal epithelial cells afterward. Hepatic progenitor cells from MSC differentiation in the growth-factor-free coculture system may contribute to the therapeutic effect for liver disease in vivo.