Open Research Projects, Research

The human assembloid reveals the crosstalk of colonic resident macrophages with their neighboring epithelium, vasculature, and enteric nervous system

Principle Investigator

Prof. Dr. Michael Sigal
Dr. Manqiang Lin

Scientific interest within the context of the graduate college:

The gastrointestinal mucosa consists of epithelial cells and a variety of surrounding stromal and immune cells. These cells act in concert to fulfill the functions of the mucosa such as nutrient absorption as well as the first line of defense against environmental threats. As one of the most prevalent immune cell types in the gut tissue, macrophages maintain mucosal barrier integrity. In addition to their role in immune defense, subsets of tissue-resident macrophages have been suggested to directly support functions of the epithelium as well as the vasculature and enteric neurons.1,2 Our research aims to understand the interplay between the epithelium, stroma, and macrophages. We would like to understand how gut resident macrophages support mucosal homeostasis and how the epithelium and stroma in turn nourish macrophages. To address this, we are now developing new organoid and assembloid models to recapitulate the cellular networks observed in vivo.

Project description:

Introduction: In the gastrointestinal mucosa, the stromal compartment has been increasingly recognized for its role in supporting epithelial proliferation and differentiation.3,4 Concurrently, the organization of stroma is also regulated by signals derived from the neighboring epithelium.5 Recently, we established an in vitro mucosa unit known as colon assembloids (Figure 1).5 This system combines adult mouse colon organoids with resident complex stromal subpopulations in a unified structure. The assembloid system faithfully recapitulates not only the anatomical morphology and cellular composition of colon epithelial crypts but also the compartmentalization of their stromal niche.5 The sophisticated self-organization of the epithelium and stroma in assembloids highlights the crucial role of their crosstalk.

Nonetheless, the current assembloid system lacks immune cells, which hinders the study of the direct interplay between the immune and other compartments. Macrophages are a critical innate immune cell type in the colon. Colonic macrophages were previously believed to originate from and to be continuously replenished by blood monocytes. However, mounting evidence suggests that gut resident macrophages may partially derive from embryonic precursors and undergo self-renewal throughout adulthood.1,6 These resident macrophages exhibit highly specialized transcriptional features, distinct localizations, and specific functions.1,7 However, their characteristics and interactions with epithelium/stroma remain poorly investigated on a functional level due to technical challenges, which is particularly true for the human mucosa.

Therefore, we aim to further develop our assembloid models by optimizing culture conditions for colon resident macrophages and adapting the technique for human cells. Once we establish an immuno-competent assembloid system that integrates resident macrophages, we will apply this system to explore the role of tissue macrophages in mucosal homeostasis.

Aim 1: Characterize the niche factors for colonic macrophages and modify the culture condition accordingly.

Aim 2: Generate human colonic assembloids containing resident macrophages and characterize their identity and spatiotemporal organization.

Aim 3: Investigate the functions of colonic macrophages in assembloids by macrophage depletion and exposure to bacterial virulence factors.

Figure 1. Cellular organization in murine colon assembloids.5 Confocal microscopy images of colon assembloids on days 1 and 4 labeled with markers for proliferative cells (KI67), colonocytes (KRT20), goblet cells (MUC2), and enteroendocrine cells (SYP). Scale bars: 100 µm.


  1. De Schepper S, Verheijden S, Aguilera-Lizarraga J, Viola MF, Boesmans W, Stakenborg N, […], Jones E, Lambrechts D, Boeckxstaens G. Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis. Cell. 2018; 175(2):400-415.e413.
  2. Fritsch SD, Sukhbaatar N, Gonzales K, Sahu A, Tran L, Vogel A, […], Pirinen E, Verhelst SHL, Weichhart T. Metabolic support by macrophages sustains colonic epithelial homeostasis. Cell Metab. 2023; 35(11):1931-1943.e1938.
  3. Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wiedenmann B, Nusse R, Amieva MR, Meyer TF. Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis. Nature. 2017; 548(7668):451-455.
  4. Kapalczynska M, Lin M, Maertzdorf J, Heuberger J, Muellerke S, Zuo X, […], Tacke F, Meyer TF, Sigal M. BMP feed-forward loop promotes terminal differentiation in gastric glands and is interrupted by H. pylori-driven inflammation. Nat Commun. 2022; 13(1):1577.
  5. Lin M, Hartl K, Heuberger J, Beccaceci G, Berger H, Li H, […], Tacke F, Rajewsky N, Sigal M. Establishment of gastrointestinal assembloids to study the interplay between epithelial crypts and their mesenchymal niche. Nat Commun. 2023; 14(1):3025.
  6. Hashimoto D, Chow A, Noizat C, Teo P, Beasley MB, Leboeuf M, […], Ginhoux F, Frenette PS, Merad M. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity. 2013; 38(4):792-804.
  7. Shaw TN, Houston SA, Wemyss K, Bridgeman HM, Barbera TA, Zangerle-Murray T, […], Allen JE, Konkel JE, Grainger JR. Tissue-resident macrophages in the intestine are long lived and defined by Tim-4 and CD4 expression. J Exp Med. 2018; 215(6):1507-1518.