Induced pluripotent stem cells (iPSCs) have self-renewal and multilineage differentiation capacities and can be used in disease modeling, drug screening, organoid construction, and cell therapy research. Directed differentiation of iPSCs depends on stage-specific signal regulation. Activin A, Noggin, DLL4, and GM-CSF respectively represent germ layer induction, BMP signaling inhibition, Notch signaling regulation, and myeloid maturation, making them typical factors for understanding iPSC differentiation protocol design.

Keywords: iPSC; induced pluripotent stem cells; Activin A; Noggin; DLL4; GM-CSF; directed differentiation; germ layer induction; BMP signaling; Notch signaling; myeloid differentiation; functional maturation

1 Cytokine Action Levels in iPSC Directed Differentiation

1.1 Understanding Cytokine Function by Differentiation Stage

iPSC directed differentiation usually involves pluripotency exit, germ layer induction, progenitor formation, lineage restriction, and functional maturation. Cytokines are used in this process to mimic embryonic development and tissue microenvironmental signals. Activin A is mainly used for early germ layer induction, Noggin regulates BMP background signaling, DLL4 mediates Notch-dependent fate decisions, and GM-CSF is mainly used for myeloid cell maturation and functional establishment. These four types of factors correspond to different regulatory levels and should not be simply understood as interchangeable “pro-differentiation factors.”

Figure 1. Directed differentiation of induced pluripotent stem cells

2 Activin A: Early Germ Layer Induction Signal

2.1 Functional Characteristics of Activin A

(1) Principle and signaling attribute

Activin A is a member of the TGF-β superfamily and has developmental regulatory significance similar to Nodal-related signaling. In iPSC differentiation, Activin A is commonly used in early induction stages to drive cells from a pluripotent state into definitive endoderm- or mesoderm-related programs. Its core function is to initiate germ layer fate, rather than directly induce the formation of mature organ cells.

(2) Applicable scenarios

Activin A is commonly used in the early stages of differentiation toward hepatocyte-like cells, pancreatic-related cells, intestinal epithelial-like cells, renal progenitors, and some cardiovascular precursors. In definitive endoderm induction, Activin A is often used to induce endoderm-related markers such as SOX17 and FOXA2. In some mesodermal or renal progenitor systems, its role needs to be interpreted together with Wnt, BMP, or FGF signaling backgrounds.

(3) Interpretation points

After Activin A treatment, differentiation success should not be judged only by early marker positivity. Definitive endoderm systems should further assess whether subsequent hepatic, pancreatic, intestinal, or other lineage markers are formed. Renal progenitor-related systems should combine PAX2, LHX1, WT1, and other markers to evaluate intermediate mesoderm and renal progenitor formation. Successful early germ layer induction is only the starting point of the differentiation process and does not mean that cells already possess mature functions.

(4) Notes

The induction effect of Activin A is strongly affected by iPSC starting state, cell density, treatment duration, and combined signaling. Unstable cell status, excessively high or low density, or an inappropriate induction window can all reduce germ layer induction efficiency. If differentiation efficiency is low, starting cell quality and treatment window should be checked first, rather than simply increasing the Activin A concentration.

2.2 Experimental Boundaries of Activin A-Related Systems

Activin A is more appropriately positioned as an early germ layer induction factor. If subsequent progenitor or mature cell formation is insufficient, the problem may not necessarily arise from Activin A itself, but may instead result from unreasonable settings of downstream BMP, FGF, Wnt, or maturation-stage signals. Activin A receptor-related indicators can help assess TGF-β/Nodal-like signal responsiveness, such as ALK-4, ACVR2A, and ACVR2B. However, receptor expression or pathway activation cannot replace target cell identity identification and still needs to be interpreted together with lineage markers and functional results.

3 Noggin: BMP Signaling Inhibition and Lineage Selection

3.1 Functional Characteristics of Noggin

(1) Principle and signaling attribute

Noggin is a classical BMP signaling inhibitor that binds BMP ligands and reduces BMP-SMAD signaling activity. In iPSC differentiation, the main function of Noggin is not to directly promote cell maturation, but to alter the lineage selection environment by inhibiting BMP background signals that are unfavorable for target lineage formation.

(2) Applicable scenarios

Noggin is commonly used in neural induction, some mesenchymal-related differentiation systems, and protocols that require reduction of non-target BMP signaling. In neural induction, BMP inhibition helps reduce interference from non-neural lineages. In some mesenchymal-like cell differentiation systems, Noggin can regulate the BMP signaling background and help cells enter the target phenotype more stably.

(3) Interpretation points

After Noggin treatment, both target lineage markers and BMP pathway activity should be observed. If target markers increase but cell status deteriorates, or if non-target lineages do not decrease, it should not be simply concluded that Noggin improves differentiation quality. A more reliable interpretation should combine BMP-SMAD signaling changes, target cell proportion, and subsequent functional maturation.

(4) Notes

Noggin is not a universal pro-differentiation factor. Some mesodermal, hematopoietic, lung epithelial, osteogenic, or chondrogenic differentiation systems require BMP signaling. Adding Noggin too early or for too long may inhibit necessary developmental signals. When using Noggin, its action stage should be clearly defined, avoiding whole-process BMP inhibition that leads to insufficient target lineage formation.

3.2 Experimental Boundaries of Noggin-Related Systems

Noggin is suitable for differentiation stages that require control of BMP background signaling. For systems that depend on BMP signaling for initiation, Noggin should be used within a specific window rather than as a long-term maintenance factor. In result interpretation, “reduced BMP signaling” should not be equated with “improved differentiation purity.” Target lineage markers, non-target lineage markers, and functional maturation indicators should be evaluated together.

4 DLL4: Notch Signaling and Fate Decision

4.1 Functional Characteristics of DLL4

(1) Principle and signaling attribute

DLL4 is an important ligand of the Notch signaling pathway and can influence cell fate decisions through contact-dependent signaling. In iPSC differentiation systems, DLL4 is commonly used to mimic Notch stimulation in hematopoietic, immune, or vascular developmental microenvironments.

(2) Applicable scenarios

DLL4 is mainly used in T cell-related differentiation, endothelial cell specification, and hematopoietic microenvironment modeling. In T cell-related systems, DLL4 can help progenitor cells enter Notch-dependent immune differentiation directions. In endothelial and vascular-related systems, DLL4/Notch signaling can participate in endothelial subtype specification and vascular structure regulation.

(3) Interpretation points

After DLL4 treatment, results should be interpreted together with Notch pathway activation and target cell function. T cell-related systems may assess markers such as CD3 and CD4/CD8 according to the target subtype; FOXP3 should be evaluated specifically in regulatory T cell-related differentiation. Endothelial-related systems should combine CD31, VE-cadherin, vWF, tube formation, and barrier function evaluation. Increased expression of a single surface marker does not indicate that cells have completed mature differentiation.

(4) Notes

DLL4 alone is usually insufficient to establish complete T cell or endothelial function. Its effect is related to presentation format, cell density, progenitor proportion, culture matrix, and supporting factors. If differentiation is unstable, DLL4 usage format, Notch activation environment, and progenitor cell quality should be checked rather than only adjusting DLL4 concentration.

4.2 Experimental Boundaries of DLL4-Related Systems

The core of DLL4-related systems is whether Notch signaling is effectively presented and whether target progenitor cells have response capacity. Soluble DLL4, immobilized DLL4, matrix-bound DLL4, or co-culture approaches may generate Notch stimulation with different intensity and duration. Therefore, experiments should focus on the correspondence among DLL4 presentation format, Notch downstream signaling, and target cell function. In T cell-related differentiation, DLL4 signaling usually needs to be combined with stage-appropriate cytokines. In endothelial-related systems, vascular markers and functional assays should be combined to judge biological significance.

5 GM-CSF: Maturation of iPSC-Derived Myeloid Cells

5.1 Functional Characteristics of GM-CSF

(1) Principle and signaling attribute

GM-CSF mainly participates in myeloid cell proliferation, survival, and functional maturation. In iPSC differentiation systems, GM-CSF usually acts during the intermediate-to-late stages after myeloid progenitor formation and is used to promote maturation and functional establishment of monocytes, macrophages, or dendritic cells.

(2) Applicable scenarios

GM-CSF is commonly used in the differentiation of iPSC-derived monocytes, macrophages, and dendritic cells. It can be combined with M-CSF, IL-3, IL-4, and other factors to regulate myeloid cell expansion, maturation, functional polarization, and inflammatory stimulus responses.

(3) Interpretation points

After GM-CSF treatment, results should be evaluated from both phenotype and function. Markers such as CD14, CD11b, and CD68 can be used to identify myeloid cell identity. Phagocytic function, inflammatory factor secretion, antigen processing capacity, or stimulus responsiveness should be used to determine whether cells have functional maturation features.

(4) Notes

Morphological changes after GM-CSF induction do not equal successful myeloid differentiation. If cells are marker-positive but functionally insufficient, common causes include low myeloid progenitor proportion, insufficient maturation duration, mismatch of supporting signals such as M-CSF or IL-4, unsuitable culture medium composition, or effects of adhesion conditions on maturation.

5.2 Experimental Boundaries of GM-CSF-Related Systems

GM-CSF is suitable for the myeloid maturation stage rather than as an early germ layer induction signal. M-CSF, IL-3, and IL-4 can serve as supporting factors in GM-CSF-related systems, but they should be selected according to the target myeloid cell type. Macrophage-like differentiation focuses more on phagocytic function and inflammatory response, whereas dendritic cell-like differentiation focuses more on antigen processing, maturation markers, and immune-stimulatory capacity. GM-CSF level changes or receptor detection can help assess system responsiveness, but final maturation still needs to be verified through myeloid markers and functional assays.

6 Differentiation Outcome Evaluation and Quality Control

6.1 Evaluation Levels of Differentiation Outcomes

The quality of iPSC-derived cells cannot rely on a single marker. Early germ layer marker expression after Activin A treatment does not mean that final organ cells are mature. Noggin-mediated reduction of BMP background does not mean that the target lineage is necessarily purified. DLL4-mediated activation of Notch-related programs does not mean that T cell or endothelial function is complete. GM-CSF-mediated myeloid maturation does not mean that cells already have stable phagocytic and inflammatory response capacity. Differentiation outcomes should be judged by combining lineage markers, functional assays, and batch stability.

6.2 Key Control Points in Differentiation Systems

6.3 Factor Activity and Storage Conditions

The activity of recombinant cytokines is affected by storage temperature, reconstitution system, freeze-thaw cycles, and batch differences. iPSC differentiation systems are sensitive to fluctuations in factor activity. The same factor may cause differences in differentiation efficiency under different batches, reconstitution methods, or storage durations. In experiments, factors should be reconstituted and aliquoted according to recommended conditions, avoiding repeated freeze-thaw cycles. When comparing different treatment groups, cell density, matrix condition, and basal culture system should be kept consistent before adjusting factor concentration and treatment window.

7 Common Problems and Result Interpretation

7.1 Low Differentiation Efficiency After Activin A Treatment

Low Activin A induction efficiency is commonly associated with iPSC starting state, cell density, induction window, Wnt/BMP background, and subsequent culture conditions. Simply increasing Activin A concentration usually cannot solve all problems and may instead increase cell death or non-target differentiation. A more reasonable approach is to first fix the starting cell state and culture density, then optimize treatment duration and supporting signals.

7.2 Target Cell Proportion Does Not Increase After Noggin Use

Noggin inhibits BMP signaling rather than directly driving maturation of all target lineages. If the target system itself requires a certain level of BMP activity, excessive Noggin may reduce differentiation efficiency. When this issue occurs, the stage of Noggin addition, treatment duration, and BMP-related signaling requirements should be reassessed.

7.3 Unstable DLL4-Related Differentiation

DLL4 mediates fate decisions through the Notch pathway, and its effect is closely related to presentation format, cell density, progenitor proportion, and co-stimulatory conditions. If T cell- or endothelial-related differentiation is unstable, DLL4 usage format, Notch activation approach, supporting factor combination, and culture microenvironment should be checked rather than only adjusting DLL4 concentration.

7.4 Insufficient Myeloid Cell Function After GM-CSF Induction

GM-CSF can promote myeloid cell maturation, but insufficient function may result from a low progenitor proportion, insufficient maturation duration, mismatch of supporting signals such as M-CSF or IL-4, or culture medium and adhesion conditions. In this case, surface markers, phagocytic function, inflammatory stimulus response, and cytokine secretion should be detected simultaneously.

8 Product Selection Related to iPSC Directed Differentiation

Activin A, Noggin, DLL4, and GM-CSF respectively correspond to germ layer induction, BMP signaling inhibition, Notch-mediated fate decision, and myeloid maturation. iPSC differentiation experiments should select factor systems according to target lineage, treatment window, and functional validation needs, and should evaluate differentiation quality through markers, pathway readouts, and functional assays.

For more related articles, please see below:

[1] Structural Features, Signaling Pathways, and Functional Differentiation Mechanisms of GDF Family Cytokines

[2] Cytokines and Inflammation

[3] The Fibroblast Development Factor (FGF) Family

[4] Regulation of TGF-beta activity by BMP-1