Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.

Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 μM) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy.

Peritoneal mesenchymal stromal cells (pMSCs) are isolated from peritoneal dialysis (PD) effluent, and treatment with the pMSCs reduces peritoneal membrane injury in rat model of PD. This study was designed to verify the identity of the pMSCs. pMSCs were grown in plastic dishes for 4-7 passages, and their cell surface phenotype was examined by staining with a panel of 242 antibodies. The positive stain of each target protein was determined by an increase in fluorescence intensity as compared with isotype controls in flow cytometrical analysis. Here, we showed that pMSCs predominantly expressed CD9, CD26, CD29, CD42a, CD44, CD46, CD47, CD49b, CD49c, CD49e, CD54, CD55, CD57, CD59, CD63, CD71, CD73, CD81, CD90, CD98, CD147, CD151, CD200, CD201, β2-micoglobulin, epithelial growth factor receptor, human leukocyte antigen (HLA) class 1, and, to a lesser extent, CD31, CD45RO, CD49a, CD49f, CD50, CD58, CD61, CD105, CD164, and CD166. These cells lacked expression of most hematopoietic markers such as CD11b, CD14, CD19, CD34, CD40, CD80, CD79, CD86, and HLA-DR. There was 38.55% difference in the expression of 83 surface proteins between bone marrow (BM)-derived MSCs and pMSCs, and 14.1% in the expression of 242 proteins between adipose tissue (AT)-derived MSCs and pMSCs. The BM-MSCs but not both AT-MSCs and pMSCs express cytokine receptors (IFNγR, TNFI/IIR, IL-1R, IL-4R, IL-6R, and IL-7R). In conclusion, pMSCs exhibited a typical cell surface phenotype of MSCs, which was not the same as on BM-MSCs or AT-MSCs, suggesting that the pMSCs may represent a different MSC lineage from peritoneal cavity.