Xiaofeng Huang

Summary: Gastric insulin-secreting organoids (GINS) represent a promising source of β-like cells for type 1 diabetes (T1D) therapy. In same-donor comparisons with induced pluripotent stem cell-derived islets (iPSC-islets), GINS displayed robust glucose responsiveness and reduced expression of key T1D autoantigens. Importantly, GINS exhibited decreased susceptibility to cytotoxicity mediated by engineered HLA-matched preproinsulin-specific effector T cells (Avatar Teffs) and a distinct transcriptional profile enriched for immune-modulatory and stress-adaptive gene programs. To enhance immune evasion, we engineered gastric stem cells to overexpress Programmed Death Ligand 1 (PD-L1) in an inducible manner. PD-L1 ⁺ GINS maintained normal functionality, while exhibiting improved survival under allogeneic Avatar Teff challenge in a MHC class I-independent fashion. We evaluated PD-L1-mediated protection against autologous Avatar Teff attack using an endothelialized microfluidic platform recapitulating physiologic immune interactions. T cells show reduced infiltration into PD-L1⁺ GINS, resulting in significantly higher organoid viability compared to control GINS. Together, these findings identify GINS as a functional and engineerable β-like cell platform with intrinsic hypoimmunogenic features, and support PD-L1 engineering as a strategy to enhance immune protection for both allogeneic and autologous transplantation in T1D.

Although specific transcription factors (TFs) are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multiomics comparison of the transcriptomes of ciliated and unciliated embryonic cells to identify TFs up-regulated in ciliated cells. We also used conditional genetics in mouse embryos and stem cells and found that the TFs SP5 and SP8 regulate cilia formation and gene expression. In embryos lacking Sp5 and Sp8 , primary and motile cilia were shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 was sufficient to induce primary cilia in unciliated cells. This work will facilitate the study of cilia assembly using stem cell models and promote further understanding of human ciliopathies.

Since its first use in 1922, insulin therapy has transformed diabetes from a fatal disease to a manageable condition. However, long-term insulin injections lead to significant complications. β-cell replacement, derived from either a limited number of deceased donors or embryonic stem cells, offers an encouraging alternative. While these procedures allow patients to be insulin-independent, they still require systemic immunosuppressants to prevent graft rejection, which poses immunological challenges. Direct reprogramming holds considerable promise as a method for generating β-cells from various sources, enabling autologous therapies that mitigate the risk of immune rejection and eliminate the need to harvest cells from embryos. This review provides an overview of the latest advances in direct reprogramming strategies, with a focus on key transcriptional regulators that drive phenotypic conversion and maintenance of various cell types into β-like cells.

While specific transcription factors are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multi-omics comparison of the transcriptomes of ciliated and non-ciliated embryonic cells to identify transcription factors upregulated in ciliated cells, and conditional genetics in mouse embryos and stem cells to demonstrate that SP5/8 regulate cilia formation and gene expression. In Sp5/8 mutant embryos primary and motile cilia are shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 is sufficient to induce primary cilia in unciliated cells. This work opens new avenues for studying cilia assembly using stem cell models and offers new insights into human ciliopathies.

Plasticity among cell lineages is a fundamental, but poorly understood, property of regenerative tissues. In the gut tube, the small intestine absorbs nutrients, whereas the colon absorbs electrolytes. In a striking display of inherent plasticity, adult colonic mucosa lacking the chromatin factor SATB2 is converted to small intestine. Using proteomics and CRISPR-Cas9 screening, we identify MTA2 as a crucial component of the molecular machinery that, together with SATB2, restrains colonic plasticity. MTA2 loss in the adult mouse colon activated lipid absorptive genes and functional lipid uptake. Mechanistically, MTA2 co-occupies DNA with HNF4A, an activating pan-intestinal transcription factor (TF), on colonic chromatin. MTA2 loss leads to HNF4A release from colonic chromatin, and accumulation on small intestinal chromatin. SATB2 similarly restrains colonic plasticity through an HNF4A-dependent mechanism. Our study provides a generalizable model of lineage plasticity in which broadly-expressed TFs are retained on tissue-specific enhancers to maintain cell identity and prevent activation of alternative lineages, and their release unleashes plasticity.

Stomach stem cells are accessible by biopsy and propagate robustly in culture, offering an invaluable resource for autologous cell therapies. Here we describe a detailed protocol to isolate, expand, engineer and differentiate human gastric stem cells (hGSCs) into pancreatic islet-like organoids containing abundant gastric insulin-secreting (GINS) cells that resemble beta-cells in molecular hallmarks and function. Sequential activation of the inducing factors NGN3 and PDX1-MAFA led hGSCs onto a novel differentiation path, including endocrine progenitor and GINS precursor, before adopting beta-cell identity, at efficiencies close to 70%. GINS organoids acquired glucose-stimulated insulin secretion in 10 days post differentiation.