Several Important In Vitro Improvements in the Amplification, Differentiation and Tracing of Fetal Liver Stem/Progenitor Cells

Wei-Hui Liu,Nan You,Hong-Bao Liu,Zheng-Cai Liu,Zhen-Bin Gong,Ke-Feng Dou,Ning Zhang,Tao Wang,Irina Kerkis

doi:10.1371/journal.pone.0047346

Abstract

ObjectiveWe previously isolated fetal liver stem/progenitor cells (FLSPCs), but there is an urgent need to properly amplify FLSPCs, effectively induce FLSPCs differentiation, and steadily trace FLSPCs for in vivo therapeutic investigation.MethodsFLSPCs were maintained in vitro as adherent culture or soft agar culture for large-scale amplification. To direct the differentiation of FLSPCs into hepatocytes, FLSPCs were randomly divided into four groups: control, 1% DMSO-treated, 20 ng/ml HGF-treated and 1% DMSO+20 ng/ml HGF-treated. To trace FLSPCs, the GFP gene was introduced into FLSPCs by liposome-mediated transfection.ResultsFor amplifying FLSPCs, the soft agar culture were more suitable than the adherent culture, because the soft agar culture obtained more homogeneous cells. These cells were with high nuclear:cytoplasmic ratio, few cell organelles, high expression of CD90.1 and CD49f, and strong alkaline phosphatase staining. For inducing FLSPCs differentiation, treatment with HGF+DMSO was most effective (P<0.05), which was strongly supported by the typical morphological change and the significant decrease of OV-6 positive cells (P<0.01). In addition, the time of indocyanine green elimination, the percentage of glycogen synthetic cells, and the expressions of ALB, G-6-P, CK-8, CK-18 and CYP450-3A1 in HGF+DMSO-treated group were higher than in any other group. For tracing FLSPCs, after the selection of stable FLSPC transfectants, GFP expression continued over successive generations.ConclusionsFLSPCs can properly self-renew in soft agar culture and effectively differentiate into hepatocyte-like cells by HGF+DMSO induction, and they can be reliably traced by GFP expression.

Highlights

The current strategies to restore liver mass and functionality are aimed at either the transplantation of hepatocyte-like cells or the stimulation of endogenous liver repair [1]
Inspired by investigations focusing on neural stem cells (NSCs) culture [16], we found that soft agar culture was well suited to amplify and maintain fetal liver stem/progenitor cells (FLSPCs)
On the first day of culturing, the cells in the soft agar culture (Figure 1A) and the cells in the adherent culture showed no difference in proliferation (Figure 1B)

Summary

Introduction

The current strategies to restore liver mass and functionality are aimed at either the transplantation of hepatocyte-like cells or the stimulation of endogenous liver repair [1]. Among the stem/progenitor cells in the treatment of liver diseases [3,4,5,6,7,8,9,10,11,12], fetal liver stem/progenitor cells (FLSPCs) are speculated to have the most therapeutic potential because they can repopulate hepatocytes and bile ducts [9,13,14]. Because FLSCPs were thought to be the smallest cells in FLCs, the cells located in the first upper layer of PCGC gradient were isolated and identified as FLSPCs. The stem characterization of FLSPCs included three aspects: detecting the expression of stem cell markers, investigating the selfrenewal ability and multiple differentiation potential. Because the isolated cells by our method achieved all the above standards, they could be identified as FLSPCs [15]

Methods

Results

Discussion

Conclusion