Bone marrow cells contribute to seven different endothelial cell populations in the heart.

Understanding the mechanisms underlying vascular regeneration in the heart is crucial for developing novel therapeutic strategies for myocardial ischemia. This study investigates the contribution of bone marrow-derived cells to endothelial cell populations in the heart, and their role in cardiac function and coronary circulation following repetitive ischemia (RI). Chimeric rats were created by transplanting BM cells from GFP female rats into irradiated male recipients. After engraftment chimeras were subjected to RI for 17 days. Vascular growth was assessed from recovery of cardiac function and increases in myocardial blood flow during LAD occlusion. After sorting GFP+ BM cells from heart and bone of Control and RI rats, single-cell RNA sequencing was implemented to determine the fate of BM cells. Our in vivo RI model demonstrated an improvement in cardiac function and myocardial blood flow after 17 days of RI with increased capillary density in the rats subjected to RI compared to Controls. Single-cell RNA sequencing of bone marrow cells isolated from rats' hearts identified distinct endothelial cell (EC) subpopulations. These ECs exhibited heterogeneous gene expression profiles and were enriched for markers of capillary, artery, lymphatic, venous, and immune ECs. Furthermore, BM-derived ECs in the RI group showed an angiogenic profile, characterized by upregulated genes associated with blood vessel development and angiogenesis. This study elucidates the heterogeneity of bone marrow-derived endothelial cells in the heart and their response to repetitive ischemia, laying the groundwork for targeting specific subpopulations for therapeutic angiogenesis in myocardial ischemia.

Protocol for generating three-dimensional induced early ameloblasts using serum-free media and growth factors.

Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta. For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Single Cell RNA Sequencing Reveals Human Tooth Type Identity and Guides In Vitro hiPSC Derived Odontoblast Differentiation (iOB).

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Application of this sci-RNA-seq analysis generates a differentiation protocol to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. We find that FGFR1c is upregulated in functional odontoblasts and specifically plays a crucial role in driving odontoblast maturity. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.

Sci-Seq of Human Fetal Salivary Tissue Introduces Human Transcriptional Paradigms and a Novel Cell Population.

Multiple pathologies and non-pathological factors can disrupt the function of the non-regenerative human salivary gland including cancer and cancer therapeutics, autoimmune diseases, infections, pharmaceutical side effects, and traumatic injury. Despite the wide range of pathologies, no therapeutic or regenerative approaches exist to address salivary gland loss, likely due to significant gaps in our understanding of salivary gland development. Moreover, identifying the tissue of origin when diagnosing salivary carcinomas requires an understanding of human fetal development. Using computational tools, we identify developmental branchpoints, a novel stem cell-like population, and key signaling pathways in the human developing salivary glands by analyzing our human fetal single-cell sequencing data. Trajectory and transcriptional analysis suggest that the earliest progenitors yield excretory duct and myoepithelial cells and a transitional population that will yield later ductal cell types. Importantly, this single-cell analysis revealed a previously undescribed population of stem cell-like cells that are derived from SD and expresses high levels of genes associated with stem cell-like function. We have observed these rare cells, not in a single niche location but dispersed within the developing duct at later developmental stages. Our studies introduce new human-specific developmental paradigms for the salivary gland and lay the groundwork for the development of translational human therapeutics.

dCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Metabolism as an early predictor of DPSCs aging.

Tissue resident adult stem cells are known to participate in tissue regeneration and repair that follows cell turnover, or injury. It has been well established that aging impedes the regeneration capabilities at the cellular level, but it is not clear if the different onset of stem cell aging between individuals can be predicted or prevented at an earlier stage. Here we studied the dental pulp stem cells (DPSCs), a population of adult stem cells that is known to participate in the repair of an injured tooth, and its properties can be affected by aging. The dental pulp from third molars of a diverse patient group were surgically extracted, generating cells that had a high percentage of mesenchymal stem cell markers CD29, CD44, CD146 and Stro1 and had the ability to differentiate into osteo/odontogenic and adipogenic lineages. Through RNA seq and qPCR analysis we identified homeobox protein, Barx1, as a marker for DPSCs. Furthermore, using high throughput transcriptomic and proteomic analysis we identified markers for DPSC populations with accelerated replicative senescence. In particular, we show that the transforming growth factor-beta (TGF-ß) pathway and the cytoskeletal proteins are upregulated in rapid aging DPSCs, indicating a loss of stem cell characteristics and spontaneous initiation of terminal differentiation. Importantly, using metabolic flux analysis, we identified a metabolic signature for the rapid aging DPSCs, prior to manifestation of senescence phenotypes. This metabolic signature therefore can be used to predict the onset of replicative senescence. Hence, the present study identifies Barx1 as a DPSCs marker and dissects the first predictive metabolic signature for DPSCs aging.