Light up neural differentiation pathways

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Stem cells have tremendous potential for use in developmental biology research, disease modeling, drug screening, and cell therapy for neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Stem cells are undifferentiated cells that have the capacity both to self-renew through mitosis and to differentiate into specialized cell types such as neuronal, liver, or muscle cells. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells (PSCs) that are commonly characterized by their expression of the transcription factors Nanog, OCT4, and SOX2, and the cell-surface proteins SSEA3, SSEA4, TRA-1-60, and TRA-1-81 (Figure 1). To verify the functional pluripotency of PSCs, they must undergo further testing to confirm their ability to differentiate into the three embryonic germ layers: ectoderm, mesoderm, and endoderm.

Mammalian neurogenesis begins with the induction of neuroectoderm, which forms the neural plate and then folds to give rise to the neural tube. These structures are composed of a layer of neuroepithelial progenitors (NEPs) that can be rapidly turned into primitive neural stem cells (NSCs). NSCs are self-renewing, multipotent progenitors present in the developing and adult mammalian central nervous system. During neural differentiation, NSCs undergo progressive lineage restrictions leading to glial progenitors (CD44+ A2B5+), which can become astrocytes (GFAP+) and oligodendrocytes (Galc+ O4+). The other branch of lineage restriction is the neuronal path leading to various types of neurons such as dopaminergic (DA) neurons (Figure 2). Table 1 provides a list of common markers and the corresponding antibodies used to characterize PSCs and NSCs (Figure 1) as well as downstream glial and neuronal cells (Figure 2).

Immunofluorescence imaging of iPSCs
Histograms of iPSCs analyzed by flow cytometry

Figure 1. Characterization of human induced pluripotent stem cells.Gibco™ Human Episomal iPSCs grown with Gibco™ Vitronectin (VTN-N) Recombinant Human Protein in Gibco™ Essential 8™ Flex Medium were stained with the indicated Thermo Scientific™ DyLight™ dye–conjugated primary antibodies and analyzed by imaging or flow cytometry. (A) Immunofluorescence imaging of iPSCs counterstained with DAPI nuclear stain (blue). Left panel: DyLight 488 anti-SSEA5 mouse monoclonal antibody (green) and DyLight 650 anti-SSEA4 mouse monoclonal antibody (red). Middle panel: DyLight 488 anti-LIN28 mouse monoclonal antibody (green). Right panel: DyLight 650 anti-SOX2 mouse monoclonal antibody (red). (B) Histograms of iPSCs analyzed by flow cytometry. Left panel: DyLight 488 mouse IgG1 isotype control antibody. Right panel: DyLight 488 anti-Nanog mouse monoclonal antibody.

Characterization of astrocytes and dopaminergic (DA) neurons derived from PSCs

Figure 2. Characterization of astrocytes and dopaminergic (DA) neurons derived from PSCs.(A) Immunofluorescence staining of glial progenitors and astrocytes generated from PD-3 iPSC-derived neural stem cells using anti-GFAP followed by Alexa Fluor™ 488 goat anti–rabbit IgG (green) antibodies and anti-CD44 followed by Alexa Fluor™ 594 goat anti–mouse IgG (red) antibodies. (B) Immunofluorescence staining of DA neurons derived from PSCs using anti–tyrosine hydroxylase followed by Alexa Fluor™ 488 donkey anti–rabbit IgG (green) antibodies. Nuclear DNA was counterstained with DAPI (blue).

Find your stem cell antibody

The characterization of stem cells is a critical step in stem cell research. No matter which detection platform you use—flow cytometry, immunocytochemistry, western blot, ELISA, or another—our collection of over 51,000 Invitrogen™ antibodies provides you with tools compatible with your experimental design.

Table 1. Selected antibodies for the characterization of stem cells and neural lineage cells. For a complete list, go to thermofisher.com/antibodies.

 TargetAntibody Cat. No. (Clone ID)
Characterization of pluripotent stem cells
Pluripotent stem cellsDNMT3BPA1-884, 49-1028
KLF4710659 (1HCLC), PA1-095
LIN28MA1-016 (14E6-4E6), MA1-016-D488 (14E6-4E6), MA1-016-D550 (14E6-4E6), MA1-016-D650 (14E6-4E6), PA1-096
NANOGMA1-017 (23D2-3C6), MA1-017-D488 (23D2-3C6), MA1-017-D550 (23D2-3C6), MA1-017-D650 (23D2-3C6), PA1-097
OCT4/POU5F1A13998 (C30A3), MA1-104 (9B7), MA1-104-D488 (9B7), MA1-104-HRP (9B7), A18525 (EM92)
PRDM14PA1-114
SALL4720030
SOX248-1400 (20G5), MA1-014 (20G5), MA1-014-D488 (20G5), MA1-014-D550 (20G5), MA1-014-D650 (20G5), PA1094
SSEA1/CD15MA1-022 (MC-480), MA1-022-D488 (MC-480), MA1-022-D550 (MC-480), MA1-022-D650 (MC-480), MA1-022-PE (MC-480), 18-0122 (MY-1), 41-1200
SSEA341-4400 (MC-631), MA1-020 (MC-631), MA1-020-D488 (MC-631), MA1-020-D650 (MC-631), MA1-020-PE (MC-631)
SSEA4MA1-021 (MC813-70), MA1-021-D488 (MC813-70), MA1-021-D550 (MC813-70), MA1-021-D650 (MC813-70), MA1-021-PE (MC813-70)
SSEA5MA1-144 (8E11), MA1-144-D488 (8E11), MA1-144-D550, MA1-144-D650 (8E11), MA1-144-D755 (8E11), MA1-144-PE (8E11)
TRA-1-60411000 (cl.A), MA1-023 (tra-1-60), MA1-023-D488X (tra-1-60), MA1-023-D550 (tra-1-60), MA1-023-D650 (tra-1-60)
TRA-1-81411100 (cl.26), MA1-024 (tra-1-81), MA1-024-D488 (tra-1-81), MA1-024-D550 (tra-1-81), MA1-024-D650 (tra-1-81)
Germ layer mesendodermBrachyury (T)MA5-17185 (1H9A2), PA5-23405
EOMESPA5-12261, MA5-24291 (644730)
GSCMA5-23070 (1C2), PA5-28380
MIXL1PA5-40323
Germ layer mesodermABCA4P21933 (3F4)
NKX2.5701622 (4H5L9), 710634 (4HCLC)
PDGFRα701142 (7H13L1), 710169 (7HCLC), PA516571, PA516742
Smooth muscle actinMA5-11544 (1A4 (asm-1)), PA5-16697, 701457 (17H19L35), 710487 (17HCLC), MA1-744 (mAbGEa)
Germ layer endodermα-Fetoprotein (AFP)710486 (9HCLC), 18-0003 (ZSA06), MA5-12754 (C3), MA5-14665 (F1-6P2A8-P2B9A9), MA5-14666 (P5B8), PA5-16658
FOXA1MA1-091 (3A8)
FOXA2701698 (9H5L7), 710730 (9HCLC), MA5-15542 (7H4B7), 720061, A16568
GATA4PA1-102
GATA6PA1-104
KLF542-3200
SOX17PA5-23352, PA5-23382
Germ layer ectodermβ-III TubulinMA1-118 (2G10)
PAX642-6600, MA1-109 (13B10-1A10)
SOX1PA5-23351, PA5-23370
Characterization of neural stem cells
Neural stem cellsNestinMA1-110 (10C2)
PAX642-6600, MA1-109 (13B10-1A10)
SOX1PA5-23351, PA5-23370
SOX248-1400 (20G5), MA1-014 (20G5), MA1-014-D488, MA1-014-D550, MA1-014-D650, MA1-014-HRP, PA1-094
Neural differentiation and characterization of glial and neuronal cells
AstrocytesGFAP13-0300 (2.2B10), MA5-12023 (ASTRO6), PA5-16291, A21282 (131-1771), A21294, A21295
Glutamine synthetase710963 (7HCLC)
S100b701340 (16H24L21), 710363 (16HCLC)
Cholinergic neuronsChATPA1-4710, PA1-4738, PA1-9027, PA1-18313, PA5-29653
DA progenitor/DA neuronsLMX1A710980 (20HCLC)
Nurr1MA1-195 (N1404), PA1-4519, PA5-13416
OTX2701948 (14H14L5), MA5-15854 (1H12C4B5), MA5-15855 (1H12G8B2), PA5-23406, PA5-29914
PITX3701181 (5H10L5), 710212 (7M5HCLC), 38-2850
Tyrosine hydroxylaseP21962, 701949, 710982

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