(K-436) Murine Colonoids and Bone Marrow Derived Macrophages as Models for Macrophage-Mediated Regenerative Response in the Intestinal Epithelium

Introduction::

The intestinal epithelium is a single-cell layer acting as a barrier against the intestine’s external environment. Over one’s lifetime, the epithelium is continually subjected to the harsh conditions within the intestinal lumen. Fortunately, intestinal tissue is the most rapidly regenerating tissue in human adults [1]. This is largely thanks to the presence of stem and progenitor cells localized in crypts [2]. Intestinal colonoids mimicking colon tissue heterogeneity and morphology can be formed from the isolation of these intestinal stem cell-containing crypts [3]. The ability of colonoids to better recapitulate features found in complex tissues allows for more intimate study of the mechanisms necessary for regeneration, placing less reliance on animal models, and providing an easily replicable system for study.

One such hallmark of intestinal regeneration is TGF-ꞵ signaling. Depending on the tissue of interest TGF-ꞵ ligands can mediate several different responses, though within the intestine, TGF-ꞵ signaling is imperative for the facilitation of an adequate regenerative response [4]. The intestine, however, is unable to synthesize the TGF-ꞵ ligands necessary (TGFꞵ1) for regeneration, and thus, receives help from macrophages, which shuttle TGFꞵ1 to damaged tissues [2]. To study macrophage-mediated regenerative response in the intestinal epithelium, a reporter sensitive to TGFꞵ1 was devised, and colonoids seeded with bone marrow derived macrophages (BMDMs) were stimulated to promote inflammation and analyze genetic response.

Materials and Methods::

Colonoids were derived from murine colonic tissue by isolation of stem cell-containing crypts. These crypts were resuspended in Cultrex^TM and seeded in organoid media enabling 3D growth and crypt formation.

BMDMs were derived from murine femur and tibia marrow. Bone marrow cells were differentiated in BMDMs by cultivation in 20 % (v/v) L929 conditioned media for 7 days. For inflammation study, colonoids (d4) were stimulated with either 0.5 % Dextran sodium sulfate (DSS) for 6 hours or 50 ng/mL Tumor necrosis factor alpha (TNFɑ) for 12 hours before adding BMDMs and Co-cultivating for 24 h further. Quantitative Real Time PCR (RT-qPCR) was conducted to track expression of genes (analyzed by 2^-ΔΔCt, n=1) possibly providing information on interactions between colonoids and BMDMs [5].

To track TGF-ꞵ signaling, a reporter construct was designed for lentiviral transduction and transfection consisting of a TGFß1 promoter and downstream reporter mNeonGreen with a destabilization domain d2EGFP.

Lentivirus carrying the TGFꞵ1 reporter was produced by HEK293T cells using the 3^rd generation lentiviral packaging system protocol [6].

Fluorescence data for transfected, using GenJet™ (SignaGen), and transduced HEK293T cells in TGFꞵ1-stimulating media was obtained using a plate-reader (Tecan, Infinite M200 Pro^TM) and confocal microscope (Leica, TCS SP5). Fluorescence in confocal images was quantified in terms of average corrected total cell fluorescence (CTCF) among all regions of interest identified using the Biovoxxel3D plugin in ImageJ.

Results, Conclusions, and Discussions::

Results: Cellular morphology confirmed successful isolation of crypt-forming colonoids and mature BMDMs. The gene expression data of inflammatory markers such as TNFα and IL-6 hint towards successful induction of inflammation by incubation with Dextran Sodium Sulfate (DSS) as well as TNFα. Responses of BMDMs to inflamed colonoids were not detected here.

The TGFß1-expression sensitive reporter was assembled correctly – confirmed by sequencing – and transfection of HEK293T cells resulted in a high rate of mNeonGreen-expressing HEK293T cells. TGFß1 responsiveness was tested by incubation with different concentrations of recombinant TGFß1. However, no significant increases in mNeonGreen levels due to 10 ng/mL or 50 ng/mL TGFß1 supplementation in the media were observed.

Discussion: RT-qPCR data mirrors results in literature confirming inflammation induction by incubation with DSS or TNFα in colonoids. High levels of activation-indicating cytokines (TNFα; IL-6) in BMDMs, independent of the presence of inflamed colonoids, might allude to strong preactivation by previous handling and explain the minor changes induced by co-culture with inflamed colonoids. Further statements would be premature due to the low repetition rates.

Conclusion: The introduction of the immune compartment to 3D organoid models increases the complexity imitable in vitro and allows the reduction of animal consumption in research. Mimicking the naturally occurring interactions between macrophages and the colonic epithelia during homeostatic and inflamed conditions would permit more detailed research in the wound healing process of colonoids. Our results suggest the need for careful handling of the BMDMs and for possibly reducing the barrier Cultrex^TM may present to the permeability of messenger substances. Studying the TGFß1 signaling activity during wound healing might call for a change in promoter (e.g., SMAD7 promoter sequence) before generating lentiviral particles and transducing colonoids to evaluate the role of TGFß1 in wound healing and coordination between immune system and epithelia [7].

The Co-cultivation of BMDMs and colonoids is a promising ex vivo model to study the complex interactions during intestinal repair.

Acknowledgements (Optional): : This research has been facilitated by the NSF International Research Experience for Students (IRES) Track I: Process Development for Cell and Tissue Biomanufacturing (Award Number: 1952614) in collaboration with Dr. Elizabeth Lipke at Auburn University and Dr. Robert Zweigerdt at Medizinische Hochschule Hannover.

References (Optional): :

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2.         Chen, L., et al., TGFB1 Induces Fetal Reprogramming and Enhances Intestinal Regeneration. bioRxiv, 2023: p. 2023.01.13.523825.

3.         Donath, S., et al., Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery. Cells, 2022. 11(7).

4.         Beck, P.L., et al., Transforming growth factor-beta mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells. Am J Pathol, 2003. 162(2): p. 597-608.

5.         Livak, K.J. and T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001. 25(4): p. 402-8.

6.         Gill, K.P. and M. Denham, Optimized Transgene Delivery Using Third-Generation Lentiviruses. Curr Protoc Mol Biol, 2020. 133(1): p. e125.

7.         Heldin, C.H. and A. Moustakas, Signaling Receptors for TGF-β Family Members. Cold Spring Harb Perspect Biol, 2016. 8(8).