Core cytokines for organoid culture: key regulatory molecules in the construction of three-dimensional organ models

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This article focuses on the core cytokines required for organoid culture, systematically elaborating the biological basis of organoids as three-dimensional structures self-organized from organ-specific cell clusters derived from pluripotent stem cells, adult stem cells, and somatic cells. It analyzes the differential functions and synergistic effects of five core cytokines—Wnt-3a, RSPO1, Noggin, EGF, and bFGF—in organoid growth, proliferation, and maintenance, and explores the application strategies of these cytokines in different organoid culture systems.

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Core Cytokines for Organoid Culture: Key Regulatory Molecules in Three-Dimensional Organ Model Construction

Brief Introduction: This article focuses on the core cytokines required for organoid culture, systematically elaborating on the biological basis of organoids as three-dimensional structures formed by self-organization of organ-specific cell clusters derived from pluripotent stem cells, adult stem cells, and somatic cells. It analyzes the differential functions and synergistic effects of five core cytokines—Wnt-3a, RSPO1, Noggin, EGF, and bFGF—in organoid growth, proliferation, and maintenance, and discusses the application strategies of these cytokines in organoid culture systems for different organs.

I. Definition and Biological Basis of Organoids.

Organoids are defined as three-dimensional structures grown from organ-specific cell clusters derived from pluripotent stem cells, adult stem cells, and somatic cells from human tissues. Organoids self-organize through cell sorting and mimic the complex structural and basic functional characteristics of various specific organs or tissues. Organoids are miniature in vitro versions of human organs, with structural and physiological similarities to the organs. Human organoids have been successfully generated for a wide range of organs, including the intestine, lung, thyroid, stomach, heart, kidney, liver, brain, and retina. These miniature organs can be stored in biobanks for basic research, such as disease modeling and developmental biology, but can also be used for translational research, such as drug screening. In addition, the potential clinical applications of organoids are multifaceted, including personalized and regenerative medicine.

From a developmental biology perspective, embryonic stem cells and induced pluripotent stem cells are important starting materials for organoid research. Somatic cells can be reprogrammed into induced pluripotent stem cells by introducing four transcription factors. As development proceeds, the blastocyst differentiates into embryonic stem cells and further forms all three germ layers: the endoderm, mesoderm, and ectoderm. The embryonic endoderm subsequently gives rise to the gastrointestinal system, heart, lung, and liver; the mesoderm develops into the kidney and vascular system; the ectoderm forms the neuroectoderm, which ultimately becomes the brain with distinct regions, including the hippocampus, optic cup, and cerebellum. Three-dimensional organoid technology can recapitulate all the above structural and functional features in vitro, and what underpins this complex self-organization process is precisely the carefully formulated combination of core cytokines in the culture medium.

II. The Core Role of Wnt-3a in Organoid Culture.

Wnt-3a is a member of the Wnt family and plays a key role in regulating multiple cellular functions, including self-renewal, proliferation, differentiation, and motility. Wnts can bind to cell membrane receptors and, by binding to adjacent cell membrane receptors, play key roles in autocrine regulation and/or paracrine modification. The signal transduction pathway mediated by Wnt genes is called the Wnt signaling pathway. Wnt-3a promotes or inhibits tumor progression through the canonical Wnt signaling pathway, depending on the cancer type. In addition, the effects of the Wnt-3a signaling pathway can be inhibited by various proteins or chemical substances. During embryonic development, Wnt-3a is necessary for normal hippocampal development, anterior-posterior patterning, somite development, and tail bud formation. Wnt-3a also promotes the self-renewal of hematopoietic stem cells, neural stem cells, and embryonic stem cells. In organoid culture, Wnt-3a is a key upstream signal for maintaining the stem cell pool of epithelial organoids such as the intestine, stomach, and liver.

III. The Enhancing Role of RSPO1 in Organoid Culture.

R-Spondin 1 is a 27 kDa secreted protein with approximately 40% identity to the other three R-Spondin family members. Injection of recombinant RSPO1 in mice leads to activation of β-catenin and proliferation of intestinal crypt epithelial cells, and ameliorates experimental colitis. As the organoid field expands, research on RSPO1 as a cell culture supplement is also increasing. RSPO1 is widely used in organoid cell culture workflows and is an important component for promoting the growth and survival of three-dimensional organoids. The core function of RSPO1 lies in stabilizing Wnt receptors on the cell membrane surface and enhancing the strength of the Wnt signaling pathway, forming a synergistic amplification effect with Wnt-3a——Wnt-3a provides the initiating signal, while RSPO1 prolongs and amplifies this signal by inhibiting the ubiquitination-mediated degradation of Wnt receptors, and together they constitute a dual safeguard for Wnt signal maintenance in organoid culture.

IV. The Differentiation-Inhibiting Role of Noggin in Organoid Culture.

Noggin can inhibit TGF-β family ligands and prevent them from binding to their corresponding receptors. Noggin was initially discovered as an antagonist of BMP-4 and was subsequently shown to regulate the activity of other BMPs (BMP-2, 7, 13, and 14). Noggin is highly conserved among vertebrates. The secreted Noggin protein regulates bone morphogenetic protein activity during development. The role of the BMP signaling antagonist Noggin in the development of osteolytic bone metastases of prostate cancer has been reported. In organoid culture, Noggin inhibits the transition of stem cells toward differentiation by blocking the BMP signaling pathway, thereby maintaining the undifferentiated state and proliferative potential of stem cells——BMP signaling promotes differentiation while Wnt signaling maintains stemness, and Noggin's blockade of BMP relieves the antagonism of BMP against Wnt signaling, making it an indispensable differentiation-inhibiting factor in long-term organoid culture.

V. The Proliferation-Promoting Role of EGF and bFGF in Organoid Culture.

Epidermal growth factor is a small growth factor containing 53 amino acid residues that can stimulate the proliferation of mesenchymal and epithelial cells. The mature protein has only 53 amino acids and is produced by proteolytic cleavage of the EGF domain near the transmembrane region. EGF and fibroblast growth factor 2 can induce the proliferation of neural precursor cells isolated from specific regions of the embryonic and adult brain. The biological activities of EGF include epithelial development, angiogenesis, inhibition of gastric acid secretion, fibroblast proliferation, and colony formation of epidermal cells in culture. bFGF is a member of the FGF superfamily of mitogenic proteins; it is a highly specific chemotactic and mitogenic factor for many cell types and appears to be involved in the remodeling of damaged tissues, such as ulcer healing, vascular repair, and traumatic brain injury. bFGF is an important component of human embryonic stem cell culture media. In addition, bFGF protein is a heparin-binding cationic protein involved in various pathological conditions, including angiogenesis and solid tumor growth. In organoid culture, EGF and bFGF provide proliferation-promoting signals for epithelial cells and mesenchymal/neural precursor cells, respectively, and are key supplementary factors for maintaining the cell expansion rate of organoids.

VI. Conclusion.

In organoid culture and Wnt signaling pathway research, high-quality recombinant RSPO1 protein is a core tool for maintaining organoid growth efficiency and experimental reproducibility. To meet the needs of organoid culture system construction and Wnt signaling pathway mechanism exploration, Uia provides R-spondin 1 Protein, Mouse. This product is suitable for applications including the culture and long-term maintenance of mouse intestinal, gastric, and hepatic organoids, research on the binding of RSPO1 to Wnt receptors and the mechanism of Wnt signal enhancement, establishment of organoid drug screening platforms, and construction of disease models related to the Wnt signaling pathway.

This article is reviewed and published by the technical expert team of UA

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