An Overview of Pluripotent and Multipotent Stem Cell Targets

Stem cells are unspecialized cells that have the capacity to self-renew and differentiate into specialized cell types, such as neurons, liver, or muscle cells. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells (PSCs) that can divide for a long period in culture. They have the potential to differentiate and become any cell type found in the human body. Adult stem cells such as neural stem cells (NSCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs) are multipotent stem cells. The differentiation of multipotent stem cells is limited to the cell types found in the tissue of origin. These cells help to maintain and repair the tissue in which they are found.

Stem cells show tremendous potential in the areas of developmental biology, disease modeling research, drug development screening, and cell therapy studies. Antibody-based detection methods such as immunocytochemistry and flow cytometry are commonly used for the characterization of various stem cells and their differentiated cells. To find more information about the different types of stem cells, stem cell markers, and antibodies we offer, please see the page contents.

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Pluripotent stem cells

Two commonly studied types of pluripotent stem cells (PSCs) are embryonic stem cells (ESCs) and induced PSCs (iPSCs). Human ESCs are isolated from the inner cell mass of the blastocyst stage of a developing embryo. iPSCs are similar or equivalent to ESCs but are generated via ectopic expression of reprogramming genes such as OCT4, KLF4, SOX2, and c-MYC in adult somatic cells. iPSCs have revolutionized stem cell research by simplifying the derivation of certain stem cells that can then be used to model diseases in a dish. These research models can be valuable in defining the mechanisms of disease pathology and can consequently play a vital role in the possible identification of therapeutic targets and drug discovery. According to research, the unlimited supply of iPSCs that can be directed to become functionally mature cells also holds great promise as source material for cell therapies that address a variety of diseases such as diabetes, liver diseases, and Parkinson’s and Alzheimer’s diseases.

Pluripotent stem cell markers

To maintain the undifferentiated stem cell state, both iPSCs and ESCs require the expression of key transcription factors such as OCT4 (figure 1) and Nanog (figure 2) that regulate genes important for cell division, differentiation, and development. In addition to these transcription factors, a specific set of cell-surface proteins such as LIN28, TRA-1-60, TRA-1-81, SSEA3, and SSEA4 are typically expressed by the PSCs. Detection of the presence of these markers is one of the first steps in characterizing newly derived iPSC and ESC lines. Specific cell staining can be achieved using antibodies against these established pluripotency markers.

Western blot analysis using anti-OCT4 recombinant antibody

Figure 1. Western blot performed on NTERA-2 (Lane 1), NCCIT (Lane 2), F9 (Lane 3), HEK-293 (Lane 4), A431 (Lane 5) and HeLa (Lane 6). Blots were probed with Anti-OCT4 Recombinant Rabbit Monoclonal Antibody (Cat. No. 701756, 1-2 µg/mL). A 38 kDa band corresponding to OCT4 was observed specifically in NTERA-2, NCCIT and F9, cell lines of embryonic origin, but not in the somatic tumor cell lines HEK-293, HeLa and A431.

Immunofluorescence analysis of Nanog using polyclonal antibody

Figure 2. Immunofluorescence analysis of Nanog was performed using NTERA-2 cells. Cells were labeled with Nanog Rabbit Polyclonal Antibody (Cat. No. PA1-097) at 5µg/mL in 0.1% BSA. (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows Nanog negative cell line HeLa with no signal.

Pluripotent stem cell differentiation

PSCs have the potential to differentiate into cells of the three embryonic germ layers: ectoderm, mesoderm, and endoderm, which can further develop into various differentiated cell types. Both iPSCs and ESCs can form embryoid bodies (EBs) when cultured in suspension in the absence of bFGF. EBs can spontaneously differentiate towards the three germ layers, which can be further developed into various differentiated cell types (figure 3). Yet another key marker for characterizing iPSCs is ESRRB (figure 4), a transcription factor that helps in self renewal and maintenance of pluripotency.

Figure 3. Pluripotent stem cell differentiation. Both iPSC and ESC can form embryoid bodies (EBs) when cultured in suspension in the absence of bFGF. EBs can spontaneous differentiate towards the three germ layers which can be further developed into various differentiated cell types.

Immunofluorescence analysis using ESRRB recombinant antibody

Figure 4. Immunofluorescence analysis of iPS cells. Cells were fixed and permeabilized for detection of endogenous ESRRB using Anti-ESRRB Recombinant Rabbit Monoclonal Antibody (Cat. No. 703237, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962), and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Cat. No. R415, 1:300) Panel a-d) shows representative control cells that were stained for detection and localization of ESRRB protein (green) with enhanced nuclear signal compared to panel e-h) showing no signal in cells differentiated to neuronal phenotype demonstrating specificity.

Differentiation of pluripotent stem cells to neural lineage cells

Neurological diseases such as Alzheimer’s, Parkinson’s, and autism, which were previously difficult to study due to a lack of in vitro cellular models, can now be studied in culture using patient-derived iPSCs. The ability to generate pluripotent stem cells from somatic cells have created an exciting new era in the field of stem cell research, particularly, neuroscience. The multipotent neural progenitor cells derived from iPSCs or ESCs are highly enriched and scalable (figure 5). They serve as an attractive alternative for primary neural stem cells or immortalized primary neurons for neuroscience research. There are several established methods to derive NSCs from iPSCs and ESCs, however, the most common one is via the inhibition of the dual SMAD pathway. The multipotent NSCs can be characterized by the presence of cellular markers such as Nestin. They can be further differentiated into lineage restricted progenitor cells. Commitment to specific neural cell types such as astrocytes, oligodendrocytes, and neurons can then be achieved by modulating the culture conditions with specific induction and maturation media. The mature lineage specified cells can be characterized by the presence of cell specific markers like MAP2, SOX10, GFAP (Figure 6, 7, 8).

Pluripotent stem cell differentiated into neural lineage cells

Figure 5. Pluripotent stem cells differentiated into neural lineage cells. Differentiation paths of PSCs to neural lineages.

Figure 6. Immunofluorescent analysis of PSD-95 (green) and MAP2 (red) on rat primary cortical neurons. Neurons were cultured for 28 days in the B-27 Plus Neuronal Culture System (Cat. No. A36534-01). At day 28 the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% triton x-100 for 30 minutes and blocked with 1% BSA for 30 minutes at room temperature. Cells were stained with anti-PSD95 antibody (Cat. No. 51-6900) at a dilution of 1:200, and anti-MAP2 (Cat. No. 13-1500) at a dilution of 1:400, in 1% BSA staining buffer, overnight at 4^oC, and then incubated with Alexa Fluor 488 conjugated donkey anti-rabbit (Cat. No. A-21206) and Alexa Fluor 594 donkey anti-mouse (Cat. No. A-21203) antibodies at a dilution of 1:1000 for 30 minutes at room temp.

Figure 7. Immunofluorescence analysis of iPSC’s differentiated to Schwann cell progenitors. Cells were fixed and permeabilized for detection of endogenous SOX10 using Anti-SOX10 Recombinant Rabbit monoclonal Antibody (Cat. No. 703439, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000). Panel b) shows representative cells that were stained for detection and localization of SOX10 protein (green) in the nucleus of Schwann cell progenitors in comparison to iPSC (a).

Figure 8. Immunofluorescent analysis of GFAP in rat cerebellum section. The rat cerebellum section was obtained following transcardial perfusion of the rat with 4% paraformaldehyde, brain was post fixed for 24 hours, and cut to 45µM. Free-floating sections were stained using an alpha internexin polyclonal antibody (Cat. No. PA1-10017) at a dilution of 1:2,000 as seen in green, using a GFAP polyclonal antibody (Cat. No. PA1-10004) at a dilution of 1:5,000 as seen in red, and with DAPI staining the nuclear DNA in blue. The alpha-internexin antibody selectively stains axons and dendrites of neuronal cells, in particular Purkinje cells and parallel fibers the axons of granule cells. The GFAP antibody labels network of glial cells, such as astrocytes in the granule cell layer and white matter and Bergmann glia in the molecular layer.

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Differentiation of pluripotent stem cells into hepatocyte-like, and pancreatic beta cells

The first step in the differentiation of hepatic and pancreatic lineage cells from PSCs is the induction of the definitive endoderm (DE). The efficiency of endoderm induction and downstream differentiation into hepatocyte like cells or pancreatic beta cells is monitored through changes in gene expression patterns and/or changes in the expression of cell surface markers assessed by immunocytochemistry and flow cytometry. During the differentiation process, PSCs gradually lose pluripotency (loss of expression of pluripotency markers such as OCT4, NANOG, SOX2, etc.) and show an exponential increase in cell size and number as they adapt to a DE fate with cells expressing mesoendodermal markers CXCR4 and GATA4 and DE markers FOXA2 and SOX17 (Figure 9). Following the DE stage, bifurcation into fore-gut endoderm and primitive gut tubes ensures further specific differentiation into hepatocyte like cells and pancreatic beta cells, respectively. Sequential differentiation strategies have been reported that allow for generation of functional glucose-responsive, insulin-secreting beta cells and hepatocyte-like cells from PSCs that have hepatic activities (Figure 10). Mature beta cells and hepatocytes can be further characterized by the presence of cellular markers such as UCN3, MAFA, C-peptide for beta-cells and AFP (Figure 11), ALB, A1AT, BSEP1, CK18, and cytochrome P450 activity for hepatocytes.

Figure 9. Immunofluorescence analysis of iPS cells and iPS cells differentiated to definitive endoderm. For immunofluorescence analysis, iPS cells and iPS cells differentiated to definitive endoderm were fixed and permeabilized for detection of endogenous SOX17 using Anti-SOX17 Recombinant Rabbit Monoclonal Antibody (Cat. No. 703063, 1:100) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Cat. No. R415, 1:300) Panel a-d) shows representative IPS cells that were stained for detection and localization of SOX17 protein (green) with no signal compared to panel e-h) clearly demonstrating enhanced nuclear localisation of SOX17 in IPS cells differentiated to definitive endoderm.

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Figure 10. Sequential differentiation of pluripotent stem cells. Schematic diagram of in vitro PSC differentiation to generate beta cells and hepatocyte-like cells.

Figure 11. Immunofluorescence analysis of AFP. Performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with AFP Mouse Monoclonal Antibody (Cat. No. MA1-19178) at 5µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d represents the merged image showing cytoplasmic and Golgi localization. Panel e represents control cells with no primary antibody to assess background.

Multipotent stem cells

Multipotent stem cells are unspecialized cells that have the ability to self-renew for long periods of time and differentiate into specialized cells with specific functions. For example, multipotent adult stem cells in bone marrow, or hematopoietic stem cells, can give rise to all blood cell types, and neural stem cells in the brain can give rise to glial and neuronal cells. However, mesenchymal stem cells can give rise to several cell types found in bone, muscle, cartilage, fat and other tissues.

Hematopoietic stem cells

Hematopoietic stem cells (HSCs) are multipotent cells that can give rise to all cell types in the blood, including B and T lymphocytes, natural killer cells, dendritic cells, monocytes, platelets, and erythrocytes (Figure 12). HSCs are very small and are non-adherent cells, which make them very difficult to purify or visualize by microscopy. In their quiescent state, HSCs can be found in the red bone marrow. Typically, HSCs are identified by flow cytometry (Figure 13) using 10-to-14 antibody panels targeting various cell-surface antigens. Recent advances in reprogramming technologies and hematopoietic differentiation from human PSCs have opened novel opportunities to study hematopoietic development and model hematological diseases.

Figure 12. Schematic diagram of hematopoietic stem cell differentiation. Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all of the cell types in the blood, including B and T lymphocytes, natural killer cells, dendritic cells, monocytes, platelets, and erythrocytes.

Figure 13. Flow cytometry of Ly-6A/E. Staining of C57Bl/6 bone marrow cells with Mouse Hematopoietic Lineage Cocktail eFluor 450 (Cat. No. 88-7772-72), Anti-Mouse CD117 (c-Kit) APC (Cat. No. 17-1171-82), and 0.125 µg of Anti-Mouse Ly-6A/E (Sca-1) PE (Cat. No. 12-5981-81). Lineage negative/low, viable cells, as determined by 7-AAD (Cat. No. 00-6993-50), were used for analysis.

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Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into many cell types, including bone, fat, cartilage, muscle, and skin (figure 14). MSCs were initially isolated from bone marrow samples, but more recently they have been found in many other types of tissue, resulting in a heterogeneous and abundant population of cells with high differentiation potential. The possible use of MSCs in treating various diseases such as cardiovascular disease and Crohn’s disease is a popular stem cell research topic.

Human MSCs have the capacity to differentiate into adipocytes (fat), chondrocytes (cartilage), and osteoblasts (bone) (Figure 15) under appropriate culture conditions. Bone marrow–derived MSCs also have the capacity to differentiate into other mesoderm-derived tissues such as myocytes (muscle cells), which express desmin, β-myosin heavy chain, α-cardiac actin, and other muscle cell markers.

Figure 14. Differentiation potential of MSCs. MSCs contribute to the stem cell niche in the marrow; contribute to smooth muscle, adipocyte, bone, and cartilage development and repair; and generally contribute to the parenchyma of most tissues and organs.

Figure 15. Immunofluorescence analysis of RUNX2. Anti-RUNX2 Recombinant Rabbit Polyclonal Antibody (Cat. No. 711519, 5 µg/mL) detects RUNX2 in nucleus of Osteoprogenitors differentiated from Bone marrow Mesenchymal stem cells. (Top row) Shows representative undifferentiated cells that were stained for RUNX2 protein (green) with no signal. (Bottom row) Clearly demonstrate the expression of RUNX2 in nucleus of differentiated osteoprogenitors specifically at day 7 and not at day 0 and 14 where mesenchymal progenitors and mature osteocytes are predominant cell population respectively.

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Neural stem cells

Mammalian neurogenesis begins with the induction of neuroectoderm, which forms a neural plate and then folds to give rise to the neural tube. These structures are made up of a layer of neuroepithelial progenitors (NEPs). NEPs can be rapidly turned into primitive neural stem cells (NSCs). NSCs are self-renewing, multipotent progenitors present in developing and adult mammalian central nervous systems. NSCs derived in vitro from pluripotent stem cells are characterized by the expression of markers such as the transcription factors POU3F2 (Figure 16), SOX1, SOX2, PAX6 (Figure 17), and the type VI intermediate filament protein nestin (Figure 18).

Figure 16. Immunofluorescence analysis of POU3F2. Neural stem cells were fixed and permeabilized for detection of endogenous POU3F2 using Anti- POU3F2 Recombinant Rabbit Polyclonal Antibody (Cat. No. 711869, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000 dilution). Panel a) shows representative cells that were stained for detection and localization of POU3F2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Cat. No. R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of POU3F2. Panel e) represents control cells with no primary antibody to assess background.

Figure 17. Immunofluorescent analysis of PAX6. Human B3 lens epithelial cells (left panel) and negative control U-2 OS cells (right panel) were fixed with formaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 1% Blocker BSA in PBS (Cat. No. 37525) for 15 minutes, all at room temperature. Cells were stained with a PAX6 monoclonal antibody (green)(Cat. No. MA1-109) in 1% Blocker BSA in PBS (Cat. No. 37525) at a dilution of 1:100 for 1 hour at room temperature, and then incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Cat. No. 35502) at a dilution of 1:250 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight-554 Phalloidin (Cat. No. 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Cat. No. 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Figure 18. Immunofluorescence analysis of endogenous Nestin. iPSC’s differentiated to Neural Rosettes were fixed and permeabilized for detection of endogenous Nestin using Anti-Nestin mouse monoclonal antibody (Cat. No. MA1-110, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27023, 1:2000). Panel b) shows representative cells that were stained for detection and localization of Nestin protein (green) in the cytoplasm of Neural Rosettes in comparison to iPSC (a).

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