Stem cells transform into a cardiac phenotype with remodeling of the nuclear transport machinery.

Nuclear transport of transcription factors is a critical step in stem cell commitment to a tissue-specific lineage. While it is recognized that nuclear pores are gatekeepers of nucleocytoplasmic exchange, it is unknown how the nuclear transport machinery becomes competent to support genetic reprogramming and cell differentiation. Here, we report the dynamics of nuclear transport factor expression and nuclear pore microanatomy during cardiac differentiation of embryonic stem cells. Cardiac progeny derived from pluripotent stem cells displayed a distinct proteomic profile characterized by the emergence of cardiac-specific proteins. This profile correlated with the nuclear translocation of cardiac transcription factors. The nuclear transport genes, including nucleoporins, importins, exportins, transportins, and Ran-related factors, were globally downregulated at the genomic level, streamlining the differentiation program underlying stem cell-derived cardiogenesis. Establishment of the cardiac molecular phenotype was associated with an increased density of nuclear pores spanning the nuclear envelope. At nanoscale resolution, individual nuclear pores exhibited conformational changes resulting in the expansion of the pore diameter and an augmented probability of conduit occupancy. Thus, embryonic stem cells undergo adaptive remodeling of the nuclear transport infrastructure associated with nuclear translocation of cardiac transcription factors and execution of the cardiogenic program, underscoring the plasticity of the nucleocytoplasmic trafficking machinery in accommodating differentiation requirements.

Simple, inexpensive method for automating tissue microarray production provides enhanced microarray reproducibility.

Tissue microarrays are a novel technology with the potential to impact cancer research by reducing the time, materials, and costs related to specimen-based marker validation. The process uses small cores of specimen tissue for molecular studies, maximizing the quantity of specimens that can be analyzed on a single slide and the results that can be obtained from a single antibody study. However, this process can be tedious and requires a significant time commitment for array production, particularly for the hand-produced tissue array blocks. In addition, this process has significant repetitive motions, risking repetitive stress injury for technical personnel. For these reasons, we have sought a simple, inexpensive system for automation of the existing microarray technologies. Using this system, slides containing as many as 400 specimens can be constructed in a simple and reproducible manner. Automation of the tissue microarray apparatus is accomplished by attaching two stepper motors to the micrometers of the apparatus that control array movement, and it has the advantages of standardizing the spacing between each specimen and eliminating repetitive motions by the user. A computer program is used to run the motors, allowing the user to input commands based on the desired moving distance. After assimilation of the motors, motor control boards, and corresponding program, the final product was tested and demonstrated to provide consistent, reproducible operation. Tissue microarrays were generated with specimen tissue diameters of 1.5 mm, 1.0 mm, and 0.6 mm with core densities upwards of 300 samples per slide.