A versatile tool for tracking the differentiation of human embryonic stem cells

doi:10.1007/s11515-010-0870-3

Front Biol

2010, Vol. 5

Issue (5) : 455-463 https://doi.org/10.1007/s11515-010-0870-3

RESEARCH ARTICLE

A versatile tool for tracking the differentiation of human embryonic stem cells

Weiqiang LI^1,2, Jie QIN^1,2, Xinyu LI³, Li ZHANG⁴, Chang LIU^1,2, Fei CHEN^1,2, Zifei WANG^1,2, Lirong ZHANG⁵, Xiuming ZHANG^1,2, Bruce T. LAHN^1,2, Andy Peng XIANG^1,2,6(

)

1. Center for Stem Cell Biology and Tissue Engineering, Sun Yat-Sen University, No. 74 Zhongshan Road 2,Guangzhou 510080, China; 2. The Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Guangzhou 510080, China; 3. Zhongshan Medical School, Sun Yat-sen University, Guangzhou 510080, China; 4. Department of Human Genetics and Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA; 5. Department of Pathophysiology, Guangdong College of Pharmacy, Guangzhou 510006, China; 6. Cell Therapy Center, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China

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Abstract

The ability of human embryonic stem cells (hESCs) to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings. However, the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult. Tissue specific promoter-driven reporter genes are powerful tools for tracking cell types of interest in differentiated cell populations. Here, we describe the construction of modular lentivectors containing different tissue-specific promoters (Tα1 of α-tubulin; aP2 of adipocyte Protein 2; and AFP of alpha fetoprotein) driving expression of humanized Renilla green fluorescent protein (hrGFP). To this end, we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation. We present a versatile method permitting target cells to be traced. Our system will facilitate research in developmental biology, transplantation, and in vivo stem cell tracking.

Keywords human embryonic stem cells lentivector transduction green fluorescent protein

Corresponding Author(s): XIANG Andy Peng,Email:xiangp@mail.sysu.edu.cn

Issue Date: 01 October 2010

Cite this article:

Weiqiang LI,Jie QIN,Xinyu LI, et al. A versatile tool for tracking the differentiation of human embryonic stem cells[J]. Front Biol, 2010, 5(5): 455-463.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-010-0870-3
https://academic.hep.com.cn/fib/EN/Y2010/V5/I5/455

Tab.1 Primers for PCR amplification

Fig.1 Structure of pLV/Final lentivectors expressing -, -, -, and -. LTR: long terminal repeat; : humanized green fluorescent protein.

Fig.2 Lentiviral transduction and characterization of H1 cells. (a) hrGFP expression in transduced H1 cells was assessed 7 days after transduction using fluorescence microscopy and fluorescence activated cell sorting (FACS). (b) After puromycin selection, a pure population of hrGFP H1 cells was obtained. Immunocytochemistry analysis showed that transduced H1 cells expressed typical embryonic stem cell markers, including alkaline phosphatase (AKP) (c), Oct4 (d), SSEA-4 (e), and TRA-1-60 (f). Bar= 100 μm

Fig.3 and differentiation of H1 cells. H1 cells that were formed fluorescent embryoid bodies (EBs) (a). Immunofluorescence analysis revealed that these transduced cells differentiated into cells of the three germ layers, expressing Nestin (b), Desmin (c), or Sox17 (d), but with maintenance of hrGFP expression. Transduced H1 cells formed fluorescent teratomas 8 weeks after implantation. Cells inside tumors were hrGFP-positive (e). Histological analysis revealed that the teratomas contained derivatives of the three germ cell layers, including ectoderm-derived neural-lineage cells (f), mesoderm-derived smooth muscle cells (g), and endoderm-derived glandular epithelium (h). Bar= 100 μm.

Fig.4 Committed differentiation of transduced H1 cells. During neural differentiation of H1 cells, hrGFP expression was first detected on day 5 (a). Expression of hrGFP increased in a time-dependent manner, and hrGFP-expressing cells with typical neuroepithelial cell characteristics were evident by day 25 (a). Immunocytochemistry showed that a small proportion of hrGFP-positive cells coexpressed Nestin (b), and that about half of such cells were βIII-tubulin-positive (c). During adipogenic differentiation of - H1 cells, hrGFP-positive cells without obvious cytoplasmic droplets first appeared on day 20 (d). About 10%-20% of hrGFP-positive cells contained small cytoplasmic droplets by day 30 (d). Oil Red O staining showed high-level lipid accumulation was inside such differentiated cells (e). During hepatic differentiation of H1 cells, some cells expressed hrGFP by day 12. Cells in genotype tended to clump and were similar in size and morphology by day 20 (f). Immunochemical analysis showed that all cells expressed alpha fetoprotein (AFP) (g). Also, differentiated cells had a high-level glycogen storage capacity, similar to that of functional hepatocytes (h). Bar= 100 μm.

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