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

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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.

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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

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Acknowledgements

The study was supported by the National Natural Science Foundation of China (NO. 30772102). We thank Fu-qin Zhang (the fourth military medical university, China) for helping us a lot in experimental design. The department of immunology (the fourth military medical university, China) helped us to do flow cytometric analyzing. Sincere thanks also go to Chun Ge (Beijing Normal University, China) for writing supports.

Disclosures

The authors indicate no potential conflicts of interest.

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Authors and Affiliations

  1. Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, 17 Changle Western Road, Xi’an, Shaanxi Province, 710032, China

    Wei-hui Liu, Ren Li & Ke-feng Dou

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  1. Wei-hui Liu
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  2. Ren Li
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Correspondence to Ke-feng Dou.

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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)

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Liu, Wh., Li, R. & Dou, Kf. Convenient and Efficient Enrichment of the CD133+ Liver Cells from Rat Fetal Liver Cells as a Source of Liver Stem/Progenitor Cells. Stem Cell Rev and Rep 7, 94–102 (2011). https://doi.org/10.1007/s12015-010-9119-4

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