Stem Cell Reviews and Reports

, Volume 7, Issue 1, pp 94–102

Convenient and Efficient Enrichment of the CD133+ Liver Cells from Rat Fetal Liver Cells as a Source of Liver Stem/Progenitor Cells

Article

Abstract

Although the stem cells are commonly isolated by FACS or MACS, they are very expensive and these is no specific marker for liver stem/progentior cells (LSPCs). This paper applied a convenient and efficient method to enrich LSPCs. The fetal liver cells (FLCs) were firstly enriched by Percoll discontinuous gradient centrifugation (PDGC) from the rat fetal liver. Then the FLCs in culture were purified to be homogeneous in size by differential trypsinization and differential adherence (DTDA). Flow cytometric analysis revealed more than half of the purified FLCs expressed alternative markers of LSPCs (CD117, c-Met, Sca-1, CD90, CD49f and CD133). In other words, the purified FLCs were heterogeneous. Therefore, they were sequentially layered into six fractions by Percoll continuous gradient centrifugation (PCGC). Both CD133 and CD49f expressed decreasingly from fraction 1 to 6. In fraction 1 and 2, about 85% FLCs expressed CD133, which were revealed to be LSPCs by high expressions of AFP and CK-19, low expressions of G-6-P and ALB. To conclude, the purity of CD133+ LSPCs enriched by combination of PDGC, DTDA and PCGC is close to that obtained by MACS. This study will greatly contribute to two important biological aspects: liver stem cells isolation and liver cell therapy.

Keywords

Fetal liver stem/progenitor cells (FLSPCs) Percoll continuous gradient centrifugation (PCGC) Percoll discontinuous gradient centrifugation (PDGC) Differential trypsinization and differential adherence (DTDA) Isolation 

Abbreviations

AFP

alpha fetoprotein

ALB

albumin

CK

cytokine

DAB

3,3′-diaminobenzidine

DTDA

differential trypsinization and differential adherence

ED

embryonic day

F1-6

fraction 1-6

FACS

fluorescence-activated cells sorting

FITC

fluorescein isothiocyanate

GGT

γ-glutamyltransferase

G-6-P

glucose-6-phosphate

HRP

horseradish peroxidase

MACS

magnetic affinity cells sorting

PCGC

Percoll continuous gradient centrifugation

PDGC

Percoll discontinuous gradient centrifugation

RT-PCR

reverse transcriptive polymerase chain reaction

Supplementary material

12015_2010_9119_Fig5_ESM.gif (10 kb)
Supplement 1

The optimal condition for gradating FLCs by PCGC. The 50% Percoll was centrifuged at 20,000×g for (A) 30, (B) 60 and (C) 90 min in angle head rotor, separately; the 40% Percoll was also centrifuged at 20,000×g for (D) 60 and (E) 90 min, respectively. Finally, the 40% Percoll centrifuged for 90 min could form continuous gradient. (GIF 9 kb)

12015_2010_9119_MOESM1_ESM.tif (1.5 mb)
High Resolution(TIFF 1564 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Hepatobiliary Surgery, Xijing HospitalFourth Military Medical UniversityXi’anChina

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