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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I1 - Engineering platforms to study BEST4+ cell differentiation

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Henry Fosnocht (he/him/his)

Undergraduate Researcher
University of North Carolina at Chapel Hill
Carrboro, North Carolina, United States

Co-Author(s)

KB

Keith Breau (he/him/his)

Graduate Student - Magness and Elston Labs
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill
Chapel Hill, North Carolina, United States
Henry H. Taylor (he/him/his)

Laboratory Technician
Magness Lab, University of North Carolina at Chapel Hill
Durham , North Carolina, United States
Joseph R. Burclaff, PhD (he/him/his)

Assistant Professor
Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University
Chapel Hill, North Carolina, United States

Primary Investigator(s)

SM

Scott Magness, Director, CGIBD Advanced Analytics Core (he/him/his)

Professor
Joint Department of Biomedical Engineering, UNC-CH/NCSU, Department of Cell & Molecular Physiology
Chapel Hill, North Carolina, United States

Introduction: The intestinal epithelium is a highly specialized layer of cells responsible for both digestion and protection of the gut. BEST4+ cells (BPCs) are a newly discovered cell type in the intestinal epithelium, only recently identified based on single cell transcriptomic studies. Very few mechanistic studies on these cells have been published, and their functions within the epithelia are largely unknown. The goal of the project is to test whether transwell and similar in vitro platforms contain BPCs as well as investigating ways to promote BPC differentiation. Literature suggests that altering culture growth kinetics or adding extrinsic factors such as using air-liquid-interface cultures, altering media pH, or changing ion content may also increase BPC differentiation. In addition, to help promote BPC differentiation, certain genetically engineered lines are being created and tested. The genes and transcription factors of interest in the genetically engineered lines are based in transcriptomic data, which has shown that two transcription factors, BARX2 and PROX1, are enriched in early BPCs and could drive differentiation. CNGA1, which is also highly expressed in BPCs, is a nonspecific divalent cation channel indicating that ions could have a roll in enriching for BPC differentiation Thus, additional aims of this project are to generate inducible overexpression lines for these genes, as well as a fluorescent BEST4-GFP-iCas9 reporter line to allow quantification and targeted ablation of BPCs in live culture.

Materials and

Methods: Experimental methods focus on differentiating primary human intestinal epithelial stem cells (hIESCs) in transwell cultures. Cells were plated onto transwells with stem cell maintenance media (MM) on both sides of the well and allowed to recover for 4-10 days, after which the cells were fed with differentiation media (DM) to induce differentiation. All cultures were monitored by light microscopy and trans-epithelial electrical resistance (TEER), which is a standard measure of epithelial barrier function. Once differentiated, cells were alternatively fixed or lysed to quantify BPC numbers by both RT-qPCR and immunofluorescent staining Electroporation of CRISPR-Cas9 protein or PiggyBAC plasmids will be used to genetically engineer PROX1 and BARX2 doxycycline inducible overexpression (iOE) DNA constructs into hIESCs. CNGA1-iOE will be expressed instead in HEK293T cells, and channel activity will be measured by patch clamping. Using the doxycycline induction system allows for the specified transcription factors to be overexpressed in an internally controlled manner. To test the impact of NOTCH signaling, the small molecule inhibitor A83-01, which we found to significantly activate NOTCH signaling in epithelial cultures, was added to the media as well as TGFB as a control.

Results, Conclusions, and Discussions: Transwell culture of wild-type (WT) primary hIESCs was first conducted to determine the normal rate of BPC differentiation in culture, demonstrating modest differentiation (Figure 1). Next, pre-differentiation culture time on transwells was tested to determine the impact of cell density on BEST4 differentiation. A strong effect was found on both the establishment of epithelial barrier integrity (TEER) and number of BPCs with a 4x increase in BEST4 RNA following an eight day incubation when compared to six day incubation (Figure 2). This indication led to testing whether this was due to changes in NOTCH signaling. The data shows that higher A83 concentration caused higher NOTCH signaling (Figure 3), but no change in BEST4 activation for the same conditions (Figure 4). In the second arm of the project, a BARX2-iOE cell line was generated with preliminary data indicating that overexpression of this transcription factor is likely insufficient to drive BPC differentiation in culture (Figure 5). More research still needs to be conducted on the BARX2 line before concluding that the BARX2 transcription factor is non-essential to BPC differentiation. Current research is focused on generating the PROX1-iOE, CNGA1-iOE line, and BEST4-GFP-iCas9 for future studies. Further experimentation will focus on testing the association of PROX1-iOE, CNGA1, and BARX2-iOE on BPC differentiation and function as well testing different formats of transwell culture such as air-liquid-interface. Additionally, experiments will be designed to adjust media ion content or pH to test if these factors play a role in BPC differentiation. Thus far, indications show that BPCs are present in transwell cultures and can be influenced using extrinsic factors, leading to a potential insight in developing a novel platform for targeted differentiation of this newly discovered cell type.

Acknowledgements (Optional): We would like to thank the anonymous organ donors, their families, and HonorBridge (formerly Carolina Donor Services), Organ Procurement Organization, Durham, North Carolina, for providing the tissue samples used in this study.