ORIGINAL ARTICLE Molecular and phenotypic characterization of CD133 and SSEA4 enriched very small embryonic-like stem cells in human cord blood A Shaikh 1 , P Nagvenkar 1 , P Pethe 1 , I Hinduja 2 and D Bhartiya 1 Very small embryonic-like stem cells (VSELs) are immature primitive cells residing in adult and fetal tissues. This study describes enrichment strategy and molecular and phenotypic characterization of human cord blood VSELs. Flow cytometry analysis revealed that a majority of VSELs (LIN - /CD45 - /CD34 + ) were present in the red blood cell (RBC) pellet after Ficoll-Hypaque centrifugation in contrast to the hematopoietic stem cells (LIN - /CD45 + /CD34 + ) in the interphase layer. Thus, after lyses of RBCs, VSELs were enriched using CD133 and SSEA4 antibodies. These enriched cells were small in size (4–6 μm), spherical, exhibited telomerase activity and expressed pluripotent stem cell (OCT4A, OCT4, SSEA4, NANOG, SOX2, REX1), primordial germ cell (STELLA, FRAGILIS) as well as primitive hematopoietic (CD133, CD34) markers at protein and transcript levels. Heterogeneity was noted among VSELs based on subtle differences in expression of various markers studied. DNA analysis and cell cycle studies revealed that a majority of enriched VSELs were diploid, non-apoptotic and in G 0 /G 1 phase, reflecting their quiescent state. VSELs also survived 5-fluorouracil treatment in vitro and treated cells entered into cell cycle. This study provides further support for the existence of pluripotent, diploid and relatively quiescent VSELs in cord blood and suggests further exploration of the subpopulations among them. Leukemia (2015) 29, 1909–1917; doi:10.1038/leu.2015.100 INTRODUCTION It was hypothesized that adult tissues may contain embryonic remnants that are ‘lost’ during developmental organogenesis that lie dormant and may give rise to some malignancies. 1 The existence of such embryonic-like cells in mouse and human tissues was demonstrated initially by Ratajczak and colleagues 2 and were termed as very small embryonic-like stem cells (VSELs). They are a population of developmentally primitive cells persisting in adult tissues. 3 They become mobilized into peripheral blood during stress situations and tissue injury, 4–7 and are enriched as LIN – CD45 – SCA1 + cell fraction in mice 2 and as LIN – CD45 – CD133 + cell fraction in humans. 8 They express pluripotent markers and have high nucleocytoplasmic ratio and undifferentiated open chromatin. 8,9 In vivo studies have demonstrated that VSELs exhibit characteristics of long‐term repopulating hematopoietic stem cells (HSCs), 10,11 are at the top of the hierarchy in the mesenchymal lineage 12 and may differentiate into organ‐specific cells. 13–16 It has also been proposed that VSELs may initiate tumor growth, 17 and recently our group has proposed a role of VSELs in ovarian 18 and pancreatic 19 cancers. However, there is still a lack of consensus on the isolation protocols for VSELs and only a handful of studies are available describing them in cord blood. 8,11,20–23 Various features of VSELs, such as their small size, pattern of expression markers or a combination of both, have been used to isolate them from cord blood. Initially, fluorescence-activated cell sorting (FACS) was employed to enrich VSELs among a population of CD34 + /LIN - / CD45 - cells. 8 This was a time-consuming protocol and not easily adapted for clinical use. The method was then improved by using a combination of red blood cell (RBC) lysis, CD133 immunomagnetic selection and FACS. 20 Aldefluor staining combined with the use of CD45 and Glycophorin A as lineage marker was also used to enrich VSELs as CD45 - /GlyA - /Aldh + cells. 24 However, these reports neither provided comprehensive cellular characterization after magnetic sorting nor have enriched VSELs using pluripotent stem cell markers like OCT4 or SSEA4. Thus, certain questions regarding properties of cord blood VSELs still remain unanswered. Attempts were made to isolate VSELs using CXCR4 and negative selection for LIN-1 and CD45. However, these studies reported absence of pluripotency in VSELs. 22,23 This inconsistency in data has led to opposing views on the presence and functionality of VSELs. 22,23,25,26 Unlike pluripotent embryonic stem (ES) and induced pluripo- tent stem (iPS) cells that divide rapidly in vitro and form teratoma in mice, 27,28 VSELs express pluripotent markers but are relatively quiescent, do not divide when cultured feeder free and do not form teratoma. 2 Quiescence in murine VSELs has been attributed to their unique genomic imprinting pattern, 9 but the quiescent nature of cord blood VSELs in terms of cell cycle status has not yet been examined. Resistance of quiescent murine bone marrow VSELs to radiotherapy 10 and testicular and ovarian VSELs to chemotherapy 29,30 has also been documented. However, similar effect of radio- or chemotherapy on human VSELs remains to be elucidated. This study aimed to enrich cord blood VSELs using cell surface markers CD133 and SSEA4 and further characterize them using various approaches. The cell cycle status and the in vitro effect of 5- Fluorouracil (5-FU, a nucleotide analog that gets incorporated into DNA and causes death of cycling cells whereas quiescent stem cells are protected 31,32 ) on cord blood VSELs has also been studied. 1 Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, India and 2 Jaslok Hospital & Research Centre, Mumbai, India. Correspondence: Professor D Bhartiya, Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai 400012, India. E-mail: deepa.bhartiya@yahoo.in or bhartiyad@nirrh.res.in Received 2 February 2015; revised 19 March 2015; accepted 7 April 2015; accepted article preview online 17 April 2015; advance online publication, 22 May 2015 Leukemia (2015) 29, 1909 – 1917 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu