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.
Similar content being viewed by others
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
References
Roberts RM, Fisher SJ. Trophoblast stem cells. Biol Reprod. 2001;84:412–21.
Khong TY, Staples A, Chan AS, Keane RJ, Wilkinson CS. Pregnancies complicated by retained placenta: sex ratio and relation to pre-eclampsia. Placenta. 1998;19:577–80.
McFadyen IR, Greenhouse P, Price AB, Geirsson RT. The relation between plasma urate and placental bed vascular adaptation to pregnancy. Br J Obstet Gynaecol. 1986;93:482–7.
Pijnenborg R, Anthony J, Davey DA, et al. Placental bed spiral arteries in the hypertensive disorders of pregnancy. Br J Obstet Gynaecol. 1991;98:648–55.
Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J. Promotion of trophoblast stem cell proliferation by FGF4. Science. 1998;282:2072–5.
Zhou S, Schuetz JD, Bunting KD, et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med. 2001;7:1028–34.
Takao T, Asanoma K, Kato K, Fukushima K, et al. Isolation and characterization of human trophoblast side-population (SP) cells in primary villous cytotrophoblasts and HTR-8/SVneo cell line. PLoS One. 2011;6:e21990.
Graham C, Hawley T, Hawley R, et al. Establishment and characterization of first trimester human trophoblast cells with expanded lifespan. Exp Cell Res. 1993;206:204–11.
Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA. 2004;101:781–6.
Xu RH, Chen X, Li DS, et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol. 2002;20:1261–4.
Amita M, Adachi K, Alexenko AP, et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc Natl Acad Sci USA. 2013;110:E1212–21.
Giakoumopoulos M, Golos TG. Embryonic stem cell-derived trophoblast differentiation: a comparative review of the biology, function, and signaling mechanisms. J Endocrinol. 2013;216:R33–45.
Niwa H, Toyooka Y, Shimosato D, et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell. 2005;123:917–29.
Keramari M, Razavi J, Ingman KA, et al. Sox2 is essential for formation of trophectoderm in the preimplantation embryo. PLoS One. 2010;5:e13952.
Desmarais JA, Demers SP, Suzuki J, et al. Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos. Reproduction. 2011;141:321–32.
McKenzie IF, Gardiner J, Cherry M, Snell GD. Lymphocyte antigens: Ly-4, Ly-6, and Ly-7. Transplant Proc. 1977;9:667–9.
Gumley TP, McKenzie IF, Sandrin MS. Tissue expression, structure and function of the murine Ly-6 family of molecules. Immunol Cell Biol. 1995;73:277–96.
Brakenhoff RH, Gerretsen M, Knippels EM, et al. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion. J Cell Biol. 1995;129:1677–89.
Inlay MA, Bhattacharya D, Sahoo D, et al. Ly6d marks the earliest stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development. Genes Dev. 2009;23:2376–81.
Quak JJ, Balm AJ, van Dongen GA, et al. A 22-kd surface antigen detected by monoclonal antibody E 48 is exclusively expressed in stratified squamous and transitional epithelia. Am J Pathol. 1990;136:191–7.
Nieuwenhuis EJ, Jaspars LH, Castelijns JA, et al. Quantitative molecular detection of minimal residual head and neck cancer in lymph node aspirates. Clin Cancer Res. 2003;9:755–61.
Kokkinos MI, Murthi P, Wafai R, Thompson EW, Newgreen DF. Cadherins in the human placenta–epithelial-mesenchymal transition (EMT) and placental development. Placenta. 2010;31:747–55.
Kusunoki S, Kato K, Tabu K, et al. The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells. Gynecol Oncol. 2013;129:598–605.
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.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13577-015-0121-7