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

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.

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.

  Target Antibody Cat. No. (Clone ID)
Characterization of pluripotent stem cells
Pluripotent stem cells DNMT3B PA1-884, 49-1028
KLF4 710659 (1HCLC), PA1-095
LIN28 MA1-016 (14E6-4E6), MA1-016-D488 (14E6-4E6), MA1-016-D550 (14E6-4E6), MA1-016-D650 (14E6-4E6), PA1-096
NANOG MA1-017 (23D2-3C6), MA1-017-D488 (23D2-3C6), MA1-017-D550 (23D2-3C6), MA1-017-D650 (23D2-3C6), PA1-097
OCT4/POU5F1 A13998 (C30A3), MA1-104 (9B7), MA1-104-D488 (9B7), MA1-104-HRP (9B7), A18525 (EM92)
PRDM14 PA1-114
SALL4 720030
SOX2 48-1400 (20G5), MA1-014 (20G5), MA1-014-D488 (20G5), MA1-014-D550 (20G5), MA1-014-D650 (20G5), PA1094
SSEA1/CD15 MA1-022 (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
SSEA3 41-4400 (MC-631), MA1-020 (MC-631), MA1-020-D488 (MC-631), MA1-020-D650 (MC-631), MA1-020-PE (MC-631)
SSEA4 MA1-021 (MC813-70), MA1-021-D488 (MC813-70), MA1-021-D550 (MC813-70), MA1-021-D650 (MC813-70), MA1-021-PE (MC813-70)
SSEA5 MA1-144 (8E11), MA1-144-D488 (8E11), MA1-144-D550, MA1-144-D650 (8E11), MA1-144-D755 (8E11), MA1-144-PE (8E11)
TRA-1-60 411000 (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-1-81 411100 (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 mesendoderm Brachyury (T) MA5-17185 (1H9A2), PA5-23405
EOMES PA5-12261, MA5-24291 (644730)
GSC MA5-23070 (1C2), PA5-28380
MIXL1 PA5-40323
Germ layer mesoderm ABCA4 P21933 (3F4)
NKX2.5 701622 (4H5L9), 710634 (4HCLC)
PDGFRα 701142 (7H13L1), 710169 (7HCLC), PA516571, PA516742
Smooth muscle actin MA5-11544 (1A4 (asm-1)), PA5-16697, 701457 (17H19L35), 710487 (17HCLC), MA1-744 (mAbGEa)
Germ layer endoderm α-Fetoprotein (AFP) 710486 (9HCLC), 18-0003 (ZSA06), MA5-12754 (C3), MA5-14665 (F1-6P2A8-P2B9A9), MA5-14666 (P5B8), PA5-16658
FOXA1 MA1-091 (3A8)
FOXA2 701698 (9H5L7), 710730 (9HCLC), MA5-15542 (7H4B7), 720061, A16568
GATA4 PA1-102
GATA6 PA1-104
KLF5 42-3200
SOX17 PA5-23352, PA5-23382
Germ layer ectoderm β-III Tubulin MA1-118 (2G10)
PAX6 42-6600, MA1-109 (13B10-1A10)
SOX1 PA5-23351, PA5-23370
Characterization of neural stem cells
Neural stem cells Nestin MA1-110 (10C2)
PAX6 42-6600, MA1-109 (13B10-1A10)
SOX1 PA5-23351, PA5-23370
SOX2 48-1400 (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
Astrocytes GFAP 13-0300 (2.2B10), MA5-12023 (ASTRO6), PA5-16291, A21282 (131-1771), A21294, A21295
Glutamine synthetase 710963 (7HCLC)
S100b 701340 (16H24L21), 710363 (16HCLC)
Cholinergic neurons ChAT PA1-4710, PA1-4738, PA1-9027, PA1-18313, PA5-29653
DA progenitor/DA neurons LMX1A 710980 (20HCLC)
Nurr1 MA1-195 (N1404), PA1-4519, PA5-13416
OTX2 701948 (14H14L5), MA5-15854 (1H12C4B5), MA5-15855 (1H12G8B2), PA5-23406, PA5-29914
PITX3 701181 (5H10L5), 710212 (7M5HCLC), 38-2850
Tyrosine hydroxylase P21962, 701949, 710982

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