Differentiation of human umbilical cord blood-mesenchymal stem cells into hepatocyte-like cells using in vitro combination of various growth differentiation factors

Zuohua CHI; Yuan ZHANG; Yan LU

ABSTRACT

BACKGROUND:

Reproductive activity of hepatocytes is limited. There are numerous studies concerning stem cells differentiation into hepatocytes, including embryonic stem cells, bone marrow cells, pancreas stem cells, neural stem cells, various sources of mesenchymal stem cells.

OBJECTIVE:

To explore the possibility of induction of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) into hepatocyte-like cells in vitro culture.DESIGN, TIME AND

SETTING:

A cytological in vitro study was performed at the Institute of Hematology, Medical College of Jinan University from October 2005 to April 2006.MATERIALS Fetus cord blood was obtained from spontaneous delivery and caesarean delivered healthy pregnant women at the Department of Obstetrics and Gynecology, First Affiliated Hospital of Jinan University. The parturients signed the informed consents.

METHODS:

UCB-MSCs were incubated in vitro, and digested in trypsin-EDTA. The third passage of cells at 5 × 10~4 cells/cm~2 wereinoculated. Original medium was removed 48 hours later. Cells were washed in phosphate-buffered saline. In the first phase, cells were incubated in F12 medium supplemented with dexamethasone, hepatocyte growth factor, epidermal growth factor and 1 ×its for 2 weeks. In the second phase, cells were incubated in F12 medium containing dexamethasone, hepatocyte growth factor,oncostatin M and 1 × ITS for two weeks.MAIN OUTCOME

MEASURES:

The following parameters were measured expression of surface marker of UCB-MSCs using flow cytometry, expression of related gene and protein of hepatocytes following induction respectively using RT-PCR and immunofluorescence staining.

RESULTS:

No CD34/CD45/CD14 of hematopoietic markers were detected, either no the CD54, CD49f, HLA-DR were found. The low expression of CD106 and high expression of CD29, CD44, CD13 were found. The gene expression of a-fetoprotein, albumin,CK-18 and TAT were discovered, and three kind of protein a-fetoprotein, albumin, CK-18 were positively observed in cytoplasm after 4 weeks of induction using immunofluorescence staining.

CONCLUSION:

UCB-MSCs are able to differentiate into hepatocyte-like cells in vitro culture following combination with many growth differentiation factors, such as dexamethasone, hepatocyte growth factor, epidermal growth factor, tumorigenesis M and ITS.