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Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo

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Abstract

The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial–mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

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Abbreviations

vSTB:

Syncytiotrophoblast

vCTB:

Cytotrophoblast

EVT:

Extravillous cytotrophoblast

EMT:

Epithelial–mesenchymal transition

TSCs:

Trophoblast stem cells

hESCs:

Human embryonic stem cells

SP:

Side-population

NSP:

Non-side population

BMP:

Bone morphogenic protein

OCT4:

Octamer-binding transcription factor4

CDX2:

Caudal type homeobox2

FBS:

Fetal bovine serum

HBM:

HTR-8/SVneo basal medium

HSM:

HTR-8/SVneo-SP medium

SOX2:

Sex determining region Y-box 2

NANOG:

Nanog homeobox

POU5F1:

Pou class 5 homeobox 1

LY6D:

Lymphocyte antigen 6 complex locus D

LY6K:

Lymphocyte antigen 6 complex locus K

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Acknowledgments

We thank Laboratory of Molecular and Biochemical Research, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan, for technical assistance. This work was in part supported by MEXT KAKENHI Grant Number 22130008 (T.T.).

Conflict of interest

There is no conflict of interest with regard to this paper.

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Author notes

  1. Kiyoko Kato

    Present address: Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Faculty of Medicine, Juntendo University, Hongo 2-1-1, Bunkyo, 113-8431, Japan

    Tetsunori Inagaki, Soshi Kusunoki, Hitomi Okabe, Izumi Yamada, Akemi Matsumoto, Shintaro Makino, Satoru Takeda & Kiyoko Kato

  2. Department of Stem Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo, 113-8510, Japan

    Kouichi Tabu & Tetsuya Taga

Authors
  1. Tetsunori Inagaki
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  2. Soshi Kusunoki
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  3. Kouichi Tabu
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Correspondence to Kiyoko Kato.

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Supplementary material 1 (DOCX 167 kb)

13577_2015_121_MOESM2_ESM.docx

Supplementary Fig. 1 MetaCore analysis between HTR-8/SVneo SP and NSP cells. We used the MetaCore package to identify the Gene GO pathway MAPs involved in upregulated genes in the microarray data on a set of HTR-8/SVneo SP cells and -NSP cells. A) Most of the upregulated genes were associated with development. B) Two Gene GO pathways of EMT were involved in the top ten scoring pathways. Supplementary Fig. 2 Cell growth of HTR-8/SVneo-SP and -NSP cells 2 x 104 SP and NSP cells were cultured with HSM medium and cell growth rate was assessed. (DOCX 202 kb)

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Inagaki, T., Kusunoki, S., Tabu, K. et al. Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo. Human Cell 29, 10–21 (2016). https://doi.org/10.1007/s13577-015-0121-7

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  • DOI: https://doi.org/10.1007/s13577-015-0121-7

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