Hormonally and chemically defined expansion conditions for organoids of biliary tree Stem Cells.

Wholly defined ex vivo expansion conditions for biliary tree stem cell (BTSC) organoids were established, consisting of a defined proliferative medium (DPM) used in combination with soft hyaluronan hydrogels. The DPM consisted of commercially available Kubota's Medium (KM), to which a set of small molecules, particular paracrine signals, and heparan sulfate (HS) were added. The small molecules used were DNA methyltransferase inhibitor (RG108), TGF- ß Type I receptor inhibitor (A83-01), adenylate cyclase activator (Forskolin), and L-type Ca2+ channel agonist (Bay K8644). A key paracrine signal proved to be R-spondin 1 (RSPO1), a secreted protein that activates Wnts. Soluble hyaluronans, 0.05 % sodium hyaluronate, were used with DPM to expand monolayer cultures. Expansion of organoids was achieved by using DPM in combination with embedding organoids in Matrigel that was replaced with a defined thiol-hyaluronan triggered with PEGDA to form a hydrogel with a rheology [G*] of less than 100 Pa. The combination is called the BTSC-Expansion-Glycogel-System (BEX-gel system) for expanding BTSCs as a monolayer or as organoids. The BTSC organoids were expanded more than 3000-fold ex vivo in the BEX-gel system within 70 days while maintaining phenotypic traits indicative of stem/progenitors. Stem-cell-patch grafting of expanded BTSC organoids was performed on the livers of Fah-/- mice with tyrosinemia and resulted in the rescue of the mice and restoration of their normal liver functions. The BEX-gel system for BTSC organoid expansion provides a strategy to generate sufficient numbers of organoids for the therapeutic treatments of liver diseases.

Single-cell transcriptome sequencing atlas of cassava tuberous root.

Introduction: Single-cell transcriptome sequencing (ScRNA-seq) has emerged as an effective method for examining cell differentiation and development. In non-model plants, it hasn't been employed very much, especially in sink organs that are abundant in secondary metabolites. Results: In this study, we sequenced the single-cell transcriptomes at two developmental phases of cassava tuberous roots using the technology known as 10x Genomics (S1, S2). In total, 14,566 cells were grouped into 15 different cell types, primarily based on the marker genes of model plants known to exist. In the pseudotime study, the cell differentiation trajectory was defined, and the difference in gene expression between the two stages on the pseudotime axis was compared. The differentiation process of the vascular tissue and cerebral tissue was identified by the trajectory. We discovered the rare cell type known as the casparian strip via the use of up-regulated genes and pseudotime analysis, and we explained how it differentiates from endodermis. The successful creation of a protoplast isolation technique for organs rich in starch was also described in our study. Discussion: Together, we created the first high-resolution single-cell transcriptome atlas of cassava tuberous roots, which made significant advancements in our understanding of how these roots differentiate and develop.