Differential Requirement of DICER1 Activity during Development of Mitral and Tricuspid Valves

Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. We specifically inactivated Dicer1 in the endocardium during cardiogenesis, and unexpectedly found that Dicer1-deletion caused congenital mitral valve stenosis and regurgitation, while it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA Sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. Our study thus reveals miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

Delivery of affordable and scalable encapsulated allogenic/autologous mesenchymal stem cells in coagulated platelet poor plasma for dental pulp regeneration

The main goal of regenerative endodontics procedures (REPs) is to revitalize teeth by the regeneration of healthy dental pulp. In this study, we evaluated the potential of combining a natural and accessible biomaterial based on Platelet Poor Plasma (PPP) as a support for dental pulp stem cells (DPSC) and umbilical cord mesenchymal stem cells (UC-MSC). A comparison study between the two cell sources revealed compatibility with the PPP based scaffold with differences noted in the proliferation and angiogenic properties in vitro. Additionally, the release of growth factors including VEGF, HGF and DMP-1, was detected in the media of cultured PPP and was enhanced by the presence of the encapsulated MSCs. Dentin-Discs from human molars were filled with PPP alone or with MSCs and implanted subcutaneously for 4 weeks in mice. Histological analysis of the MSC-PPP implants revealed a newly formed dentin-like structure evidenced by the expression of Dentin sialophosphoprotein (DSPP). Finally, DPSC induced more vessel formation around the dental discs. This study provides evidence of a cost-effective, xenofree scaffold that is compatible with either autologous or allogenic strategy for dental pulp regeneration. This attempt if successfully implemented, could make REPs treatment widely accessible, contributing in improving global health conditions.

Transdifferentiation Meets Next-generation Biotechnologies

Transdifferentiation is the process of converting terminally differentiated cells to another cell type. Being less time-consuming and free from tumorigenesis, it is a promising alternative to directed differentiation, which provides cell sources for tissue regeneration therapy and disease modeling. In the past decades, transdifferentiation was found to happen within or across the cell lineages, being induced by overexpression of key transcription factors, chemical cocktail treatments, etc. Implementing next-generation biotechnologies, such as genome editing tools and scRNA-seq, improves current protocols and has the potential to facilitate discovery in new pathways of transdifferentiation, which will accelerate its application in clinical use.

Investigating the in vivo biodistribution of extracellular vesicles isolated from various human cell sources using positron emission tomography

Noninvasive imaging is a powerful tool for understanding the in vivo behavior of drug delivery systems and successfully translating promising platforms into the clinic. Extracellular vesicles (EVs), nano-sized vesicles with a lipid bilayer produced by nearly all cell types, are emerging platforms for drug delivery. To date, the biodistribution of EVs has been insufficiently investigated, particularly using nuclear imaging-based modalities such as positron emission tomography (PET). Herein, we developed positron-emitting radiotracers to investigate the biodistribution of EVs isolated from various human cell sources using PET imaging. Chelator conjugation did not impact EVs size and subsequent radiolabeling was found to be highly efficient and stable with Zr-89 (t1/2 = 78.4 h). In vivo tracking of EVs isolated from bone marrow-derived mesenchymal stromal cells (BMSCs EVs), primary human macrophages (Mϕ EVs), and a melanoma cell line (A375 EVs) were performed in immunocompetent ICR mice. Imaging studies revealed excellent in vivo circulation for all EVs, with a half-life of approximately 12 h. Significantly higher liver uptake was observed for Mϕ EVs, evidencing the tissue tropism of EV and highlighting the importance of carefully choosing EVs cell sources for drug delivery applications. Conversely, the liver, spleen, and lung uptake of the BMSC EVs and A375 EVs was relatively low. We also investigated the impact of immunodeficiency on the biodistribution of BMSC EVs using NSG mice. The spleen uptake drastically increased in NSG mice, which could confound results of therapeutic studies employing this mouse models. Lastly, PET imaging studies in a melanoma tumor model demonstrated efficient tumor uptake of BMSC EVs following intravenous injection. Overall, these imaging studies evidenced the potential of EVs as carriers to treat a variety of diseases, such as cancer or in regenerative medicine applications, and the necessity to understand EVs tropism to optimize their therapeutic deployment.

Dissecting the Complexity of Early Heart Progenitor Cells

Early heart development depends on the coordinated participation of heterogeneous cellsources. As pioneer work from Adriana C. Gittenberger-de Groot demonstrated, characterizing thesedistinct cell sources helps us to understand congenital heart defects. Despite decades of researchon the segregation of lineages that form the primitive heart tube, we are far from understanding itsfull complexity. Currently, single-cell approaches are providing an unprecedented level of detail oncellular heterogeneity, offering new opportunities to decipher its functional role. In this review, wewill focus on three key aspects of early heart morphogenesis: First, the segregation of myocardial andendocardial lineages, which yields an early lineage diversification in cardiac development; second,the signaling cues driving differentiation in these progenitor cells; and third, the transcriptionalheterogeneity of cardiomyocyte progenitors of the primitive heart tube. Finally, we discuss howsingle-cell transcriptomics and epigenomics, together with live imaging and functional analyses, willlikely transform the way we delve into the complexity of cardiac development and its links withcongenital defects.

Cross-Tissue Characterization of Heterogeneities of Mesenchymal Stem Cells and Their Differentiation Potentials

Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative medicine and the treatment of autoimmune disorders. Comparing MSCs from different tissues at the single-cell level is fundamental for optimizing clinical applications. Here we analyzed single-cell RNA-seq data of MSCs from four tissues, namely umbilical cord, bone marrow, synovial tissue, and adipose tissue. We identified three major cell subpopulations, namely osteo-MSCs, chondro-MSCs, and adipo/myo-MSCs, across all MSC samples. MSCs from the umbilical cord exhibited the highest immunosuppression, potentially indicating it is the best immune modulator for autoimmune diseases. MSC subpopulations, with different subtypes and tissue sources, showed pronounced differences in differentiation potentials. After we compared the cell subpopulations and cell status pre-and-post chondrogenesis induction, osteogenesis induction, and adipogenesis induction, respectively, we found MSC subpopulations expanded and differentiated when their subtypes consist with induction directions, while the other subpopulations shrank. We identified the genes and transcription factors underlying each induction at the single-cell level and subpopulation level, providing better targets for improving induction efficiency.

New Frontiers towards Regeneration of the Intervertebral Disc: On Progenitor Cells, Growth Factors and Biomaterials

This Special Issue on intervertebral disc (IVD) regeneration focuses on novel advances in understanding the cell sources and culture conditions of various cell types, i.e., progenitor and IVD cells. The issue consists of seven articles that provide a comprehensive overview of recently applied research insights: (1) into how IVD herniation can be provoked in a controlled in vitro biomechanical testing set-up, (2) how cells can be used for IVD repair, (3) the physiological conditions of IVD cells and (4) how hyaluronic acid could be used for IVD repair, and (5) how nucleus pulposus progenitor cells (NPPCs) and mesenchymal stromal cells (MSCs) shall be cultured and expanded towards a possible cell therapy.

Colonic healing requires WNT produced by epithelium as well as Tagln+ and Acta2+ stromal cells

While Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Wnt blockage in distinct stromal cell types suggested obligatory functions of several stromal cell sources and yielded different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affected colonic Reg4+ epithelial cell differentiation, and impaired colonic epithelial regeneration after injury. Single cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin alpha 2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that Wnt blockage from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated Wnt blockage from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.

Higher Chondrogenic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells Compared to Chondrocytes-EVs In Vitro

The inability of cartilage to self-repair necessitates an effective therapeutic approach to restore damaged tissues. Extracellular vesicles (EVs) are attractive options because of their roles in cellular communication and tissue repair where they regulate the cellular processes of proliferation, differentiation, and recruitment. However, it is a challenge to determine the relevant cell sources for isolation of EVs with high chondrogenic potential. The current study aims to evaluate the chondrogenic potential of EVs derived from chondrocytes (Cho-EV) and mesenchymal stem cells (MSC-EV). The EVs were separately isolated from conditioned media of both rabbit bone marrow MSCs and chondrocyte cultures. The isolated vesicles were assessed in terms of size, morphology, and surface marker expression. The chondrogenic potential of MSCs in the presence of different concentrations of EVs (50, 100, and 150 μg/ml) was evaluated during 21 days, and chondrogenic surface marker expressions were checked by qRT-PCR and histologic assays. The extracted vesicles had a spherical morphology and a size of 44.25 ± 8.89 nm for Cho-EVs and 112.1 ± 10.10 nm for MSC-EVs. Both groups expressed the EV-specific surface markers CD9 and CD81. Higher expression of chondrogenic specified markers, especially collagen type II (COL II), and secretion of glycosaminoglycans (GAGs) and proteoglycans were observed in MSCs treated with 50 and 100 μg/ml MSC-EVs compared to the Cho-EVs. The results from the use of EVs, particularly MSC-EVs, with high chondrogenic ability will provide a basis for developing therapeutic agents for cartilage repair.

MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains

Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.