Where new approaches can stem from: Focus on stem cell identification


Stem cells are among the key players in regenerative medicine and stem cell therapy, and thus they are the keen focus of cell biologists' interest. Unequivocal identification of specific stem cell types is the prerequisite for their purification by cell sorting and for follow-up of therapeutic regenerative strategies.

The first stem cells to be identified were hematopoietic stem cells (HSCs). This was over four decades ago. Their key functional characteristics-self renewal and multilineage differentiation—have proved to be ubiquitous for all adult tissue—or organ-derived stem cells, and even for pluripotent embryonic, nuclear transfer, and induced pluripotent stem cells. Although the use of embryonic stem cells for research and therapeutic purposes raises various legal and ethical issues, and nuclear transfer and induced pluripotent stem cells are not yet characterized thoroughly enough to be used for such purposes, the adult human body appears to host in most of its tissues stem cells that can be extracted, propagated in vitro, and used for therapeutic purposes. In fact, these resident stem cells probably have as their biological role the maintenance and regeneration of the given tissue throughout the life of the individual. To fulfil this role, resident stem cells need to exhibit some specific properties in addition to the ability of self renewal, which is achieved by keeping at least one of the daughter cells as a stem cell during mitosis. Primarily, they need to be in an undifferentiated state, but possess high differentiation potential—implying the ability to differentiate into all cell types of their home tissue and possibly into other cell types as well, when appropriate (experimental) circumstances are provided. Furthermore, stem cells are a population of cells that are in a growth arrested state most of the time, but can re-enter the cell-cycle on demand-for example upon tissue injury-and give rise to a differentiating and highly proliferative progeny. These latter are termed progenitor cells or transient amplifying cells, and are very similar to stem cells (including some of their molecular markers) in most aspects, except they do not renew themselves and thus their population becomes terminally differentiated after a limited, though sometimes enormous number of cell divisions. In conjunction with slow cycling, stem cells require a stem cell niche: a microenvironment that provides external factors necessary for maintaining stem cell properties and functions, collectively often referred to as “stemness.” Although not a consensus yet, stem and progenitor cells are sometimes together termed precursors, indicating that both can be expanded under appropriate conditions.

The discovery of some stem cells, like that of HSCs has come through observing the normal and pathological differentiation processes of ever-renewing tissues of the body and these have, therefore, easily assimilated into our views of cellular evolution. We have gained vast knowledge in the field of hematopoietic stem cells and this area is probably the most established field of stem cell research. The state of the art in identifying hematopoietic stem cells is summarized in this issue by Challen et al. (1).

The discovery of yet other stem cells, such as neural stem cells came as a great surprise. Clear evidence for their existence goes back to the early 80′s when Goldman and Nottebohm (2) confirmed adult neurogenesis during the singing season of songbirds. Reynolds and Weiss (3) succeeded in establishing cultures of neural stem cells and induced them to neural differentiation, thereby overturning the long-lasting dogma that no new neurons are generated postnatally. Since their discovery, neural stem cells isolated from the embryonic or adult brain have turned into powerful tools for the study of molecular mechanism of neurogenesis (4) with applications for cell regeneration therapies in neurological disorders. In this issue, the most recently evolving perspectives of neuronal stem cell differentiation are discussed (5).

Unlike in the case of pluripotent embryonic stem cells, several adult stem and progenitor cell types can give rise only to a limited number of differentiated cell types. However, 2 years ago a unique adult pluripotent stem cell population, the very small embryonic-like stem cells (VSELs) were discovered in murine bone marrow (6) and have consequently become the subject of investigation in various other murine and human tissues as well. They have now been detected in several adult organs of the mouse and the data being covered in a recent Rapid Publication of the journal (7).They are further characterized in this focus issue in the context of other “small stem cell” populations described in the literature (8).

Although VSELs promise almost as broad potential as embryonic stem cells do, they also pose a challenge inherent in their pluripotency. Stem cells of other origins appear to be somewhat further down the line of differentiation and thus appear more amenable to applications in regenerative therapies. The daily exposure of the cornea to physical, chemical, and biological impacts often lead to deteriorated vision and the requirement to restore both its transparency and functional metabolism, which is made difficult by its complex histological structure. Fortunately, in the past couple of years our knowledge about the vastly different (hypothetical) stem/progenitor cells of the three distinct tissue layers of the cornea has greatly expanded. In this issue, the potential molecular markers emerging through high density array technology, as well as isolation and expansion methods are reviewed in detail (9).

Given that among the leading causes of death those of cardiovascular origin amount to over half of the total, the potential role of endothelial progenitor cells in regenerative treatment is extremely important. In this issue, the role of these progenitor cells in maintaining and restoring vascular endothelial function, as well as their liberation to form the bone marrow and molecular characterization is reviewed (10).

Unfortunately, stem cells can also turn against their host body: the term cancer stem cell has recently invaded our research nomenclature. Are they generated from normal stem cells? Why would these undergo mutations if they hardly ever need to divide in their stem cell niche? Do they evolve by ordinary (bulk) cancer cells further climbing back on the path of de-differentiation? It appears that we get many answers to the many systems we examine. One thing is not debated though: cancer stem cells appear most resistant to therapy and can easily serve to regenerate their original tumors, giving rise to relapses of the disease. The controversies of cancer stem cell theory and clonal evolution, as well as the current approaches for identifying these cells that “Die Hard” are also discussed in this issue (11).

Overall, the six reviews in this issue focus on characterization, isolation, and enrichment of various stem/progenitor cells. These characterizations go beyond several highlighted manuscripts in the past, as those often focused on single antigens and their expression in a mixed population. By contrast, in the present issue special attention is devoted to the combination of a variety of phenotypic and functional markers that are needed to identify specifically different stem cell types. With this in mind, we aimed to summarize the most important molecular markers presented in this focus issue (Table 1). This table shows that classical stem cell markers like CD34 or CD133 are almost ubiquitously expressed on stem cells from various sources, whereas these cells differ greatly in terms of cell/tissue specific markers. We hope that new ideas and approaches shall stem from this collection of markers, and the set of reviews presented in this special issue may help many to specifically isolate the cell type of interest and exploit it for therapeutic purposes.

Table 1. Compilation of characteristic positive and negative surface antigen and intracellular markers detectable by flow cytometry for very small embryonic-like stem cells (VSELs), neural stem cells (NSC), hematopoietic stem cells (HSCs), limbal epithelial stem cells (LESCs), endothelial progenitor cells (EPCs) and cancer stem cells (CSC)
Cell typeVery small embryonic-like stem cells (vSELs)Neural stem cells (NSC)Hematopoietic stem cells (HSCs)Limbal epithelial stem cells (LESCs)Endothelial progenitor cells (EPCs)Cancer stem cells (CSC)a
  •  Data are summarized from Refs.1,5, and8–11. +, marker expressed; −, not expressed; empty fields, not tested. Lineage markers: Mac-1, Gr-1, CD4, CD8, CD45R/B220, and TER119.

  • a

    CSC markers are not specific for all types of CSC but depend on cell type.

  • b

    May not be an exclusive marker.

  • c

    Human VSELs.

  • d

    Hoechst exclusion is not restricted to stem cells, most progenitor cells and non-stem cancer cells can also possess ABC transporters.

  • e

    Average value for murine and human VSELs, respectively.

ABCG2   +b  
Bmi-1   +b  
C/EBPδ   +b  
CD11b (MAC-1)    
CD20  +
CD44   +
CD81 +    
CD95 +   
CD105   + 
CD117 (C-kit) +  +
CD118 (LIF-R)      
CD133+c+ ++
CD144 (Ve-Cadherin)    + 
CD184 (CXCR4)+c   + 
Lineage markers  
Notch-1 +    
Sca-1 (mouse)+ +   
VEGF-R2    + 
ΔNp63α   +b  
Hoechst side  populationd+/− ++ +
Size3.63; 6.58 μme  10.0–16.0 μm