Identification of Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Circulating in Peripheral Blood During Organ/Tissue Injuries

Abstract

We have identified in adult tissues a population of pluripotent very small embryonic/epiblast-like stem cells (VSELs) that we hypothesize are deposited at onset of gastrulation in developing tissues and play an important role as backup population of tissue-specific/committed stem cells. We envision that during steady-state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. VSELs similarly as epiblast-derived migrating primordial germ cells change the epigenetic signature of some of the imprinted genes and therefore remain quiescent in adult tissues. These epigenetic changes in methylation status of imprinted genes prevent them also from teratoma formation. Mounting evidence indicates that VSELs are mobilized into peripheral blood during tissue/organ injuries and enumeration of these cells may be of prognostic value (e.g., in stroke or heart infarct). In this chapter, we will present FACS-based strategies to detect and enumerate these cells in human peripheral blood and umbilical cord blood.

Introduction

The rapidly developing regenerative medicine is searching for safe and therapeutically efficient sources of stem cells (SCs) that should give rise to cells from all three germ layers and could be employed to regenerate damaged organs/tissues. SCs endowed with such broad spectrum of differentiation are described during embryogenesis and are called pluripotent stem cells (PSCs) (Ratajczak et al., 2007a). Our group identified and isolated a population of pluripotent Sca-1⁺Lin⁻CD45⁻ very small embryonic-like stem cells (VSELs) from adult murine bone marrow (BM), murine fetal livers (FLs), and several adult murine organs including brain, liver, kidney, lungs, skeletal muscles, and retina (Kucia et al., 2006a, Liu et al., 2009, Zuba-Surma et al., 2008d, Zuba-Surma et al., 2009b). These cells express several morphological (e.g., relatively large nuclei containing euchromatin) and molecular (e.g., expression of SSEA-1, Oct4, Nanog, Rex1) markers characteristic for embryonic SCs (ESCs) or epiblast SCs (EpiSCs). We hypothesize that VSELs are deposited during early gastrulation in developing tissues/organs, survive into adulthood, and play an important role as a backup population of PSCs in the turnover of tissue committed stem cells (TCSCs) (Kucia et al., 2007b, Ratajczak et al., 2007a).

However, the existence of PSCs in adult tissues had been postulated by several investigators; such cells were never purified and identified at the single-cell level. Thus, the presence of pluripotent VSELs in adult tissues may reconcile previously published data stating that adult tissues may contain a population of PSCs (Ratajczak et al., 2007a, Ratajczak et al., 2007b). The presence of such cells with multilineage differentiation capabilities was postulated mainly based on experiments showing that some populations of cells isolated from adult tissues enriched among adherent cells may contain primitive cells that differentiate into various tissues. Such cells were defined as either (i) mesenchymal stem cells (MSCs); (ii) multipotent adult progenitor cells (MAPCs); (iii) marrow-isolated adult multilineage inducible (MIAMI) cells; (iv) multipotent adult stem cells (MASCs); and (v) OmniCytes (Beltrami et al., 2007, D’Ippolito et al., 2004, Jiang et al., 2002, Radcliff and Jaroszeski, 1998). It is conceivable that all these cells are closely related, overlapping populations of SCs described by different investigators and given various names according to circumstance. The potential relationship between these cells and VSELs is not clear at this moment. However, since all these cells are largely derived from the adherent fraction of BM- or adult organ-derived cells, they could as we envision potentially contain from beginning some VSELs.

Similar population of CD133⁺Lin⁻CD45⁻ cells was identified in human umbilical cord blood (UCB), mobilized peripheral blood (PB), and adult BM (Kucia et al., 2007a, Zuba-Surma et al., 2010). Table I summarizes the most important features of murine and human VSELs. We noticed that the number of these circulating cells increases both in mice and in humans during stress situations related to tissue organ injuries (e.g., heart infarct, stroke, acute colitis) as well as after administration of certain drugs employed in hematology to mobilize hematopoietic stem progenitor cells (HSPCs) into PB (Abdel-Latif et al., 2010, Kucia et al., 2008, Paczkowska et al., 2009, Wojakowski et al., 2006, Wojakowski et al., 2009, Zuba-Surma et al., 2008b).

We noticed that when purified murine VSELs are plated over a C2C12 myoblast feeder layer, they form spheres that resemble embryoid bodies (EBs) (Kucia et al., 2006a). The VSEL-derived spheres (VSEL-DSs) contain primitive stem cells that, after replating into tissue differentiation-specific media, differentiate into cells from all three germ layers. Furthermore, while we observed that freshly isolated VSELs do not exhibit in vitro and in vivo hematopoietic potential, after coculture over OP9 stromal cells they can differentiate along the hematopoietic lineage in a similar way as ESCs or induced pluripotent stem cells (iPSCs). Cells derived from these OP9-primed VSELs acquire expression of several hemato/lymphopoiesis-specific genes and markers, give rise to hematopoietic colonies in vitro, and protect lethally irradiated mice in both primary and secondary transplant models upon transplantation (Zuba-Surma et al., 2008c, Zuba-Surma et al., 2009a). We also observed that, compared to hematopoietic stem/progenitor cells (HSCs), VSELs are highly resistant to irradiation. Based on these observations, we postulate that VSELs are the most primitive murine BM-residing population of stem cells that have the potential to become specified into the hematopoietic lineage and thus may share some of the characteristics of long-term repopulating HSCs (Zuba-Surma et al., 2008c, Zuba-Surma et al., 2009a). Thus, BM-residing VSELs nicely support a concept that these cells are precursors of TCSCs – in this particular case they are precursors of HSPCs.

We also reported that VSELs are mobilized into PB during organ injuries (e.g., heart infarct, stroke), which suggests that these cells could participate in the regeneration of damaged tissues (Abdel-Latif et al., 2010, Kucia et al., 2006b, Paczkowska et al., 2009, Wojakowski et al., 2006, Wojakowski et al., 2009, Zuba-Surma et al., 2008b). In this chapter, we will discuss cytometry-based methods to detect and to enumerate VSELs circulating in PB. We already noticed that enumeration of these cells may be also of clinical/prognostic value in patients after heart infarct or stroke (Abdel-Latif et al., 2010, Paczkowska et al., 2009, Wojakowski et al., 2006, Wojakowski et al., 2009). More studies, however, are needed to support these observations.

Identification of circulating VSELs requires unique gating strategies to focus on small events that are slightly smaller than red blood cells (RBCs) (Ratajczak et al., 2009, Zuba-Surma and Ratajczak, 2010). Furthermore, since both small size and density VSELs are lost (up to 50%) during Ficoll-Paque centrifugation (Zuba-Surma et al., 2010), the recommended way to preserve these cells is lysis of PB samples to preserve VSELs for staining and subsequent analysis.