BackgroundThe biological basis for sex differences in brain function and disease susceptibility is poorly understood. Examining the role of gonadal hormones in brain sexual differentiation may provide important information about sex differences in neural health and development. Permanent masculinization of brain structure, function, and disease is induced by testosterone prenatally in males, but the possible mediation of these effects by long-term changes in the epigenome is poorly understood.MethodsWe investigated the organizational effects of testosterone on the DNA methylome and transcriptome in two sexually dimorphic forebrain regions—the bed nucleus of the stria terminalis/preoptic area and the striatum. To study the contribution of testosterone to both the establishment and persistence of sex differences in DNA methylation, we performed genome-wide surveys in male, female, and female mice given testosterone on the day of birth. Methylation was assessed during the perinatal window for testosterone's organizational effects and in adulthood.ResultsThe short-term effect of testosterone exposure was relatively modest. However, in adult animals the number of genes whose methylation was altered had increased by 20-fold. Furthermore, we found that in adulthood, methylation at a substantial number of sexually dimorphic CpG sites was masculinized in response to neonatal testosterone exposure. Consistent with this, testosterone's effect on gene expression in the striatum was more apparent in adulthood.ConclusionTaken together, our data imply that the organizational effects of testosterone on the brain methylome and transcriptome are dramatic and late-emerging. Our findings offer important insights into the long-term molecular effects of early-life hormonal exposure.

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This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)‐diacrylate, poly(ethylene glycol)‐fibrinogen, and gelatin methacrylate. Cell‐laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale‐up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time‐consuming and costly. Using a new molding technique, the microfluidic device employs a modified T‐junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10–60 million cells mL−1) and a wide range of diameters (300–1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long‐term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor‐based cell expansion and differentiation, and high throughput tissue sphere‐based drug testing assays.

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A common challenge in cell therapy is the inability to routinely maintain survival and localization of injected therapeutic cells. Delivering cells by direct injection increases the flexibility of clinical applications, but may cause low cell viability and retention rates due to the high shear forces in the needle and mechanical wash out. In this study, we encapsulated endothelial colony forming cells (ECFCs) in poly(ethylene glycol)-fibrinogen (PF) hydrogel microspheres using a custom-built microfluidic device; this system supports rapid encapsulation of high cell concentrations (10 million cells per mL) and resulting cell-laden microspheres are highly uniform in shape and size. The encapsulated ECFCs were shown to have >95% viability and continued to rapidly proliferate. Expression of cell markers (von Willebrand factor, CD105, and CD14), the ability to form tubules on basement membrane matrix, and the ability to take up low-density lipoprotein were similar between pre- and post-encapsulated cells. Viability of encapsulated ECFCs was maintained after shear through 18-23-gauge needles. Ex vivo and in vivo cell delivery studies were performed by encapsulating and injecting autologous equine ECFCs subcutaneously into distal limb full-thickness wounds of adult horses. Injected ECFCs were visualized by labeling with fluorescent nanodots before encapsulation. One week after injection, confocal microscopy analysis of biopsies of the leading edges of the wounds showed that the encapsulated ECFCs migrated into the surrounding host tissue indicating successful retention and survival of the delivered ECFCs. Rapid, scalable cell encapsulation into PF microspheres was demonstrated to be practical for use in large animal cell therapy and is a clinically relevant method to maintain cell retention and survival after local injection.