Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.

Summary. Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high-resolution procedure for tracking 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE-fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that the addition of Flt3-ligand (FL) to a cytokine cocktail consisting of Steel factor, IL-3, IL-6 and G-CSF increased the proportion of CD34, human marrow cells initially recruited into division in vitro, shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34 þ CD38 ¹ population through several (up to four) cell generations. These studies also showed that during the first 4 d there was no detectable apoptosis among the progeny of the CD34 þ (CD45RA/CD71) ¹ cells generated in the presence of this four-cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.

Development of microneedles for unskilled and painless collection of blood or drug delivery addresses the quality of healthcare through early intervention at point-of-care. Microneedles with submicron to millimeter features have been fabricated from materials such as metals, silicon, and polymers by subtractive machining or etching. However, to date, large-scale manufacture of hollow microneedles has been limited by the cost and complexity of microfabrication techniques. This paper reports a novel manufacturing method that may overcome the complexity of hollow microneedle fabrication. Prototype microneedles with open microfluidic channels are fabricated by laser stereolithography. Thermoplastic replicas are manufactured from these templates by soft-embossing with high fidelity at submicron resolution. The manufacturing advantages are (a) direct printing from computer-aided design (CAD) drawing without the constraints imposed by subtractive machining or etching processes, (b) high-fidelity replication of prototype geometries with multiple reuses of elastomeric molds, (c) shorter manufacturing time compared to three-dimensional stereolithography, and (d) integration of microneedles with open-channel microfluidics. Future work will address development of open-channel microfluidics for drug delivery, fluid sampling and analysis.