Circulating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

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Endogenous mechanisms underlying the remodeling of neuronal circuitry after mammalian CNS injury or disease remain primarily unknown. Here, we investigated axonal plasticity after optic nerve injury and found that macrophages recruited into the injury site and adult retinal ganglion cell (RGC) axons, which undergo injury-induced sprouting and terminal remodeling, were linked by their respective expression of a ligand and receptor pair active in axon guidance. Recruited macrophages specifically upregulated mRNA encoding the guidance molecule EphB3 and expressed EphB proteins capable of binding Ephrin B molecules in vivo and in vitro. Injured adult RGC axons in turn expressed EphrinB3, a known receptor for EphB3, and RGC axons bound recombinant EphB3 protein injected into the optic nerve. In vitro, EphB3 supported adult RGC axon outgrowth, and axons turned toward a source of this guidance molecule. In vivo, both reduction of EphB3 function in adult heterozygous animals and loss of function in homozygous animals greatly decreased RGC axon re-extension or sprouting after optic nerve injury. Comparisons of axon re-extension in EphB3 null and wild-type littermates showed that this loss of axonal plasticity was not attributable to a difference in intrinsic axon growth potential. Rather, the results indicated an essential role for local optic nerve-derived EphB3 in regulating adult RGC axon plasticity after optic nerve injury. Of note, the loss of EphB3 did not affect the ability of injured RGC axons to elaborate complex terminal branching, suggesting that additional EphB3-independent mechanisms governed adult axon branching triggered by CNS damage.