Harnessing the Power of Wnt3a: Promoting Growth and Development of Organoids

Organoids are a three-dimensional model established in vitro that is highly similar to tissues or organs in vivo, and can reproduce the complex spatial morphology of differentiated tissues and the interactions between various cells. They play an important role in the fields of disease modeling, organ regeneration and personalized therapy. As a more physiologically relevant model, organoid models are increasingly used in cancer research, regenerative medicine, drug discovery, and broader organoid research. The culture of organoids requires the maintenance of various cytokines, among which Wnt3a is one of the most commonly used factors, occupying a key position in the culture of most tissue-derived organoids. This role has made Wnt3a highly relevant for organoid culture, organoid technology, and advanced culture systems designed for research use.

Wnt3a is a member of the Wnt protein family, a wnt family of structurally related genes that encode secreted signaling proteins. The wnt gene family consists of multiple members, and Wnt3a, also known as Wnt family member 3a or Wnt family member 3a, is a protein coding gene whose wnt gene product functions as a secreted signaling protein in the canonical wnt signaling pathway. This signaling pathway is central to embryonic development, several developmental processes, normal embryonic mesoderm development, and normal morphogenesis. Wnt3a can bind transmembrane receptors, including Frizzled and related co-receptors, which are often described as seven transmembrane receptors in the Wnt signaling pathway, so that β-catenin can accumulate in the cell and bind to the nucleus The combination of transcription factors LEF/TCF initiates the expression of downstream target genes such as c-Myc and Cyclin D1, and then participates in the regulation of cell development, proliferation, differentiation, adhesion, polarity, intercellular communication, survival and self-renewal, etc. In addition, Wnt3a can also only bind Frizzled to activate the non-canonical Wnt pathway. Through this signaling pathway, Wnt3a influences cellular components linked to tissue specific functions, organ development, and skeletal maturation.

Figure 1. Wnt3a signaling pathway [1]. 

Wnt3a can not only promote the growth, proliferation and differentiation of normal cells, but also participate in the regulation of cell fate and pattern during tumorigenesis and embryonic development. Its functions are also studied in developmental biology, tissue engineering, and tissue culture systems involving pluripotent stem cells, human pluripotent stem cells, induced pluripotent cells, embryonic stem cells, adult stem cells, and other stem cell populations. Based on these important functions, Wnt3a as a cytokine has been widely used in primary cell culture research. It is also relevant in cell culture workflows involving different cell types, including tumor cells, t cells, and specialized cultures supported by extracellular matrix components and growth factors.

Wnt3a Promotes Organoid Growth

Wnt3a has been shown to be an important component for maintaining the proliferation of Lgr5+ stem cells in intestinal epithelial cells. Sato T et al. found that in the presence of substances such as Wnt3a, a single Lgr5+ cell derived from intestinal crypts could be cultured into intestinal organoids [2]. This finding supports the idea that Wnt3a mediates self renewal while allowing cells to expand in a physiologically relevant model that better reflects in vivo tissue architecture. In the high-content Wnt3a medium, most of the cells in the intestinal crypt area were EdU-positive, showing obvious proliferation characteristics, and no differentiation was seen. When Wnt3a was withdrawn, the cells stopped proliferating and differentiated [3].

Figure 2. Wnt3a promotes crypt-villous unit generation in the small intestine. (Top: Without Wnt3a; Bottom: With Wnt3a) [3]. 

In addition, studies by Bartfeld S. et al. have shown that adding Wnt3a can significantly promote the formation of gastric organoids without karyotype changes [4]. In the establishment of organoid models derived from gastric cancer tissue, higher concentrations of Wnt3a are more effective than lower concentrations [5]. This has direct relevance for tumor organoids and patient derived organoids used in disease modeling and drug discovery.

Figure 3. Wnt3a promotes proliferation of gastric organoids [4]. 

Wnt3a has been used in the culture of various organoids. In addition to the intestinal and gastric organoids described above, it can also be used in the culture of organoids such as pancreas, liver, thyroid, esophagus and nervous system. Current organoid culture applications also include liver organoids, kidney organoids, brain organoids, and intestinal organoids, alongside emerging systems for adult lung research and other organ development studies. These culture systems provide a more physiologically relevant model for studying developmental processes, therapeutic agents, and disease mechanisms that may not be captured well in conventional animal models.

In recent years, the field of organoids has continued to heat up. In order to support the development of organoid culture technology, KACTUS has successfully developed a series of organoid cytokine products such as recombinant Wnt3a with high activity and low endotoxicity. These products are designed to support organoid technology, cell culture optimization, and broader research use in stem cell and tissue engineering applications. The performance is stable and reliable, and can be applied to the cultivation of various organoids and applied research, and at the same time provide stem cells and immune cells and other primary cell culture-related factor products to meet customer needs.

KACTUS Wnt3a can successfully induce the activity of the Topflash reporter gene in HEK293T cells.

KACTUS recombinant Wnt3a protein can successfully induce the activity of the Topflash reporter gene in HEK293T cells, with an ED50 value of 21.02 ng/mL, which is significantly better than competing products. Its high activity may benefit applications that require stable Wnt3a signaling in extracellular domain-driven receptor interactions, including studies linked to fzd protein biology, gene ontology annotations, and body fluids or matrix-supported culture conditions involving extracellular matrix components.

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References

[1] Volleman TNE, Schol J, Morita K, Sakai D, Watanabe M. Wnt3a and wnt5a as Potential Chondrogenic Stimulators for Nucleus Pulposus Cell Induction: A Comprehensive Review. Neurospine. 2020 Mar;17(1):19-35.

[2] Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD, Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011 Nov;141(5):1762-72.

[3] In JG, Foulke-Abel J, Estes MK, Zachos NC, Kovbasnjuk O, Donowitz M. Human mini-guts: new insights into intestinal physiology and host-pathogen interactions. Nat Rev Gastroenterol Hepatol. 2016 Nov;13(11):633-642.

[4] Bartfeld S, Bayram T, van de Wetering M, Huch M, Begthel H, Kujala P, Vries R, Peters PJ, Clevers H. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology. 2015 Jan;148(1):126-136.e6.

[5] Wuputra K, Ku CC, Kato K, Wu DC, Saito S, Yokoyama KK. Translational models of 3-D organoids and cancer stem cells in gastric cancer research. Stem Cell Res Ther. 2021 Sep 6;12(1):492.