Matthew Barron

Expression of retinol binding protein and transthyretin during early embryogenesis

Previous studies have shown that anterior lateral plate endoderm from stage 6 chicken embryos is necessary and sufficient to enable precardiac mesoderm to complete its cardiogenic program in vitro, culminating in a rhythmically contractile multicellular vesicle (Sugi and Lough [1994] Dev. Dyn. 200:155–162). To identify cardiogenic factors, we have begun to characterize proteins that are secreted by endoderm cell explants. Fluorography of proteins from endoderm‐conditioned medium revealed 1–2 dozen bands, the most prominent of which migrated at approximately 17 and 25 kD. The bulk of the 17‐kD band, which migrates near FGFs and subunits of the transforming growth factor‐β family, was identified by N‐terminal sequencing as transthyretin (TTR). A component of the 25‐kD band was identified by Western blotting as retinol binding protein (RBP). RT/PCR analysis revealed that mRNAs for both proteins are in the embryo as early as stage 3. In situ hybridization localized these mRNAs to the extraembryonic endoderm at stage 6, after which they were detected in endoderm overlying the embryo proper, including the developing heart. Later, RBP and TTR mRNA and protein were detected in cells associated with the developing heart. Western blotting of whole embryo proteins revealed the presence of RBP by stage 7, followed by sequential increases to stage 25; by contrast, content of RBP in isolated hearts peaked at stage 14, then declined. Immunohistochemistry revealed the presence of RBP protein in the extracellular matrix subjacent to lateral plate endoderm beginning at stage 8; upon formation of the definitive heart, intense staining was observed in the cardiac “jelly.” By contrast TTR was intracellular, first detected as subtle deposits in stage 6 embryonic endoderm, which by stage 8 were prominent in the dorsally invaginated endoderm subjacent to the precardiac splanchnic mesoderm. At stages 11–14, TTR was detected only in myocardial cells. Such localization of RBP and TTR may indicate a role in the transport and distribution of retinol and thyroid hormone, respectively, from yolk to embryo prior to establishment of the circulatory system, and is suggestive of a subsequent role in heart development. Dev. Dyn. 1998;212:413–422.

Making stem cells infarct avid.

A major factor limiting the engraftment of transplanted stem cells after myocardial infarction is the low rate of retention in the infarcted site. Our long-term objective is to improve engraftment by enabling stem cells to recognize and bind infarcted tissue. To this end, we proposed to modify the surface of embryonic stem cells (ESCs) with the C2A domain of synaptotagmin I; this allows the engineered stem cells to bind to dead and dying cardiac cells by recognizing phosphatidylserine (PS). The latter is a molecular marker for apoptotic and necrotic cells. The C2A domain of synaptotagmin I, which binds PS with high affinity and specificity, was attached to the surface of mouse ESCs using the biotin-avidin coupling mechanism. Binding of C2A-ESCs to dead and dying cardiomyocytes was tested in vitro. After the surface modification, cellular physiology was examined for viability, pluripotency, and differentiation potential. C2A covalently attached to the ESC surface at an average of about 1 million C2A molecules per cell under mild conjugation reaction conditions. C2A-ESCs avidly bound to dying, but not viable, cardiomyocytes in culture. The normal physiology of C2A-modified ESCs was maintained. The binding of C2A-ESCs to moribund cardiomyocytes demonstrates that the retention of transplanted cells may be improved by conferring these cells with the ability to bind infarcted tissue. Once established, this novel approach may be applicable to other types of transplanted therapeutic cells.

Rapid single-step separation of pluripotent mouse embryonic stem cells from mouse feeder fibroblasts.

Highly enriched, pure populations of pluripotent mouse embryonic stem (mES) cells are a prerequisite to downstream experimental manipulations. However, the existing preplating method does not allow complete removal of co-cultured mouse embryonic fibroblast (MEF) feeder cells. The primary objective of the current investigation was to develop and validate a rapid, single-step separation technique for the complete removal of MEF feeder cells from mES cells. A discontinuous density gradient was prepared using Histopaque 1119 at incremental percentages from the top to bottom of a test tube (20, 40, 60, and 100% in culture medium). A suspension of mES cells and MEF feeder cells was layered on top of the gradient. After centrifugation at 400 x g, ES cells and MEF feeder cells were segregated discretely in separate layers at the 40/20% and 100/60% density interfaces, respectively. The mES cells were enriched to a purity of greater than 99% with a recovery rate of greater than 90%. The separation did not alter the viability or the differentiation potential of mES cells. This study validates a simple technique that enables the preparation of highly enriched mES cells that are essentially free of contaminating MEF feeder cells. The discontinuous density gradient separation method is inexpensive, efficient, rapid, and reproducible. The method can be readily scaled-up to accommodate large batch preparations, enabling a broad range of processing needs. Overall, this simple technique significantly expedites the recovery and enrichment of mES cells from MEFs.