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Stem Cells Biomarkers

Rosaria Esposito
Tags: Stem Cells

Different types of stem cells

There has been at least one moment in everyone’s life in which the motivational sentence “You can be whatever you want to be” was particularly true, and that was in the few days immediately following the lucky gametes encounter that led to our life.
The concepts of “potential” and “stemness” are so strictly related that, when thinking to “stem cells”, we tend to consider primarily the embryonic ones (ESCs), as they are the ultimate example of “stemness”. In fact, in the early moments of our existence we were totipotent cells: the zygote and the cells derived from its first divisions have the potential to form all cell types required to develop the embryo and the extraembryonic supporting structures.
After a few division cycles, these cells undergo a first restriction of their fate becoming pluripotent: the blastocyst’s inner cell mass can differentiate into all the tissues of the body, but not the placenta, which derives instead from the outer cell mass. Embryonic stem cells are considered multipotent when they specialize into endoderm, mesoderm and ectoderm, the three germ layers each giving rise to specific differentiated tissues of the fetus and, later on, of the adult organism (Zakrzewski et al. Stem Cell Research & Therapy, 2019).

However, even if the embryonic stem cells are a great example of the different degrees of “potency”, these are not the only one.

In the adult organism small quantities of stem cells can be found throughout the body in specific niches, the microenvironment where they reside and, most importantly, receive stimuli that determine their fate (Ferraro et al., Adv Exp Med Biol, 2010). They are usually referred to as progenitor cells or somatic (or adult) stem cells and are responsible for the growth, maintenance and repair of the tissues they belong to. Therefore, they can normally differentiate only into a few cell types and are considered multipotent, oligopotent, and even uni-/bi- potent. For example, the multipotent Hematopoietic Stem Cells give rise to both the lymphoid and myeloid blood lineages and, in turn, the myeloid and the lymphoid oligopotent precursors will give rise only to a few specific cell types each. Moving to different tissues, example of unipotent progenitors could be the spermatagonial stem cells, only capable of developing into sperm cells.

The growing interest of research work in the stem cells field, led in 2006 to the “invention” of the induced Pluripotent Stem Cells (iPSC). These cells derive from adult somatic cell, genetically reconverted to an embryonic stem cell-like fate via the expression of four “Yamanaka Factors”: Oct3/4, Sox2, c-Myc, and Klf4 (Takahashi & Yamanaka, Cell, 2006). iPSCs are pluripotent as they have the ability to differentiate into cells from all three germs layers, and are similar to ESC also in self-renewal, morphology, growth kinetics properties (Los et al., Stem Cells and Biomaterials for Reg Med, 2019). During the past decade, iPSC technology has become an attractive in vitro model to generate clinically relevant cells, including neurons, cardio-myocytes, and hepatocytes. Important applications of iPSC include the study of human development, modeling disease and the development of cell-based therapies in regenerative medicine. They are extremely valuable in the biomedical research field as they circumvent the major limitations of the natural stem cells: since they derive from adult somatic cells, there are no ethic concerns regarding their manipulation; in addition, they can be obtained with no major limitations in the starting material.

Different levels of stem cells potency

Figure 1. Different levels of stem cells potency

Stem cell markers

Identification and isolation of stem cells rely on specific molecular signatures and surface antigen markers. Given the high heterogeneity and complexity underlying the “stem cells” populations, due to their origin, location, differentiation state, species, the search for new markers is not an easy task, and the list continues to expand. The paragraph below will describe a few of the most commonly used stem cell markers, divided into two main groups depending on their function and cellular localization: transcription factors and cell surface markers. It is also worth noticing here that, in general, the same common pluripotency markers are used to identify embryonic stem cells (ESCs) and iPSCs, given their high degree of similarity.

Transcription factors. They represent the core of the network responsible for the embryonic stem cells features, regulating the stemness pathways. So far, approximately 25 of them have been identified. The better known examples are probably the components of the Yamanaka “reprogramming cocktail” (Oct-3/4, c-Myc, Sox2, Klf4), as well as Nanog, all involved in stem cell self-renewal and pluripotency maintenance (W. Zhato et al., Cancer Transl Med, 2017). Among the transcription factors less often cited, but still playing a role in stem cells characterization, we could mention: LEF1/TCF1, acting downstream the Wnt signaling; the ESC Associated Transcript (ECAT) genes, acting at different levels in stem cells maintenance, proliferation and differentiation; the Developmental Pluripotency-associated (DPPA) Genes, a group of five proteins related by names only, described as a set of Oct4-related genes, serving as markers for early embryonic and germline pluripotent cells (Zhao et al., Molecules, 2012). Interestingly some markers, such as Rex1, highly expressed in ESCs, do not seem to have a functional significance in pluripotency maintenance or embryo development (Zhao et al., Molecules, 2012).

Cell surface markers. This class is a particularly efficient tool for pluripotent cells characterization and isolation, notably via cell sorting, given the accessibility of these molecules to the antibodies used to this aim. Among the most commonly used there are the Stage Specific Embryonic Antigens (SSEA-1, -3, -4), a group of carbohydrate-associated molecules controlling cell-cell communication and interaction with intracellular structures during development (A. Bruce et al, BioEssays, 1990). In particular SSEA-3 and SSEA-4 are considered markers for human ESCs/iPSC, whereas SSEA-1 is a ESCs marker in mouse. TRA-1-60 and TRA-1-81, two antigens recognized by the homonymous antibodies, are also popular pluripotency markers even though the exact molecular identities of these epitopes has not been fully elucidated for a long time. It is now known that they correspond to keratan sulfate-related structures, expressed on pluripotent cell surface and suggested to be associated to podocalyxin (Badock et al., Cancer Res, 1999; Schopperle and DeWolf, Stem Cells, 2007; Natunen et al., Glycobiology, 2011).
Additional cell surface markers are often shared with adult stem cells. For example CD133 is a common hematopoietic stem cell marker. The Stem Cell Factor (SCF), a cytokine existing both as a transmembrane protein and a soluble protein, support the survival of stem cells and is involved in the development of hematopoietic, gonadal and pigment cell lineages. The integrins protein family mediate the attachment of the cell to the surrounding tissues, playing therefore an important role in the formation of the stem cells niches. In mammals, there are 24 known heterodimeric integrin receptors, each with specific binding partners and different roles.

Adult Stem cell markers

As previously mentioned, some undifferentiated adult stem cells (aka tissue stem cells), responsible for organ and tissues homeostasis and repair, are present throughout the human body. They can be hard to isolate and propagate in culture. Studying these populations is fundamental to increase our general knowledge about the regulatory mechanisms underlying their biological activity, their response to aging, injury, or pathological conditions. The identification of suitable characterizing markers is therefore fundamental to facilitate research activities. Hematopoietic Stem Cells (HSCs, also known as Blood Stem Cells), the Mesenchymal Stem Cells (MSCs), and the Neural Stem Cells (NSCs) are all examples of adult stem cells populations.

Hematopoietic Stem Cells Markers. Hematopoietic Stem Cells can differentiate into all blood cell types, including erythrocytes, platelets, as well as lymphocytes, granulocytes, and macrophages. These cells are mainly present in adult bone marrow, but they can also reside in the umbilical cord blood and a small amount of peripheral blood. HSCs are often isolated using panels of antibodies, as opposed to a single universal marker.
CD34 is among the most common HSC markers, useful to isolate this small population from the vast majority of mature blood cells. Since it is known that CD34+ cells also include lineage-committed progenitor cells, other markers can be used in parallel to distinguish the actual multipotent Long-Term HSCs (self-renewing population) from the multipotent progenitors (MPPs or Short-Term HSCs) only able to sustain hematopoiesis for a short period. An example of these additional markers is CD90 (or Thy-1), a glycoprotein also expressed on other cell types, such as the MSCs. Despite the popularity of CD34+, several studies demonstrated that some CD34- cells display stemness features, and that the CD34+ population could in fact derive from a CD34- early progenitor (Osawa et al., Science, 1996; Bhatis et al., Nat. Med Science, 1998; Zanjani et al. Exp. Hematol., 1998; Nakamura et al. Blood, 1999). Therefore the selection of CD34+ cells could exclude a part of the primitive hematopoietic stem cells.
For this reason, ABCG2 (ATP-binding cassette superfamily G member 2) has been proposed as an alternative marker. ABCG2 is a member of the ABC transporters, a family of proteins responsible for the translocation of different substrates across the cell membrane. ABCG2 is expressed in the so-called Side Population (SP) and it is actually responsible for its characterizing feature: the ability to efflux the Hoechst dye, thus displaying a much lower staining compared to other bone marrow cell types. The SP is enriched in HSC, and most of the SP cells bear other typical HSCs surface markers (Camargo et al., Blood, 2006). ABCG2 can be detected in CD34- negative cells, and it is then downregulated when CD34 starts to be expressed (Zhou et al. Nat. Med., 2001).
Finally, another classic way to identify the HSC is the characterization of the KLS phenotype: the population of cells c-Kit+, Lin- and Sca1+ is considered enriched in HSC.

Mesenchymal Stem Cells Markers. These cells were discovered in the bone marrow, but they can actually be isolated from different sources, such as the umbilical cord, the adipose tissues, the dental pulp and the synovium. They are defined as: 1) cells adherent to plastic surfaces in standard culture conditions; 2) capable of self-renewal and differentiating into bone, adipose and cartilage tissue in culture; 3) expressing specific cell surface markers. Since MSCs can derive from different tissues, the specific markers they express can also be different. Example of markers common to mesenchymal stem cells from different sources (i.e. bone marrow, peripheral blood, adipose tissues, dental pulp) are CD90 and CD105.
It is worth mentioning the antigen Stro-1, deriving its name from the first monoclonal antibody used to isolate MSCs from the bone marrow, and considered the historical key marker for these cells. Even though much of the characterization of MSCs and their precursors can in fact be attributed to the Stro-1 antibody, the molecular nature of this antigen has not being fully characterized, and only recently it has been reported as heat shock cognate 70 (HSC70; Fitter et al., Stem Cells. 2017).

Neural Stem Cells Markers. NSCs are the self-renewing, uncommitted progenitors of the nervous system, giving rise to a diversity of neural lineages, encompassing neurons, astrocytes, and oligodendrocytes. They are therefore interesting candidates for cell-based therapies in neurodegenerative disorders, such as Parkinson's disease or multiple sclerosis. The most common NSCs marker is Nestin, an intermediate filament of class VI, transiently expressed in these cells.

Musashi-1 (Msi1) is also widely used to label NSCs. It belongs to an evolutionarily conserved family of RNA-binding proteins and it is involved in the maintenance of the proliferation of multipotential neural stem/progenitor cells. Finally, other previously mentioned general stem cell markers, such as Sox2, CD133, ABCG2, can also be used to identify NSCs.

Enzo Life Sciences offers a comprehensive Stem Cell research portfolio. At Enzo, we believe in providing scientists with novel tools that will help stem cell discovery. We have our SCREEN- WELL® Stem Cell Library, a collection of 130 compounds that can be used for stem cell signalling research and drug screening. We also offer a broad range of antibodies for use in all areas of stem cell research, including very efficient antibodies against the Yamanaka factors Oct3/4 or c-Myc. Every antibody is backed by our Worry-Free Antibody Trial program, allowing you to evaluate any of our antibodies of interest for your specific application or species without risk. Furthermore, our protein portfolio encompasses a variety of growth factors to differentiate your choice of cells. Do not hesitate to contact our Technical support team if you need any kind of assistance!

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