An engineered cardiac reporter cell line identifies human embryonic stem cell-derived myocardial precursors.

Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease

Antibody-drug conjugate targeting CD46 eliminates multiple myeloma cells

Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q

High-Throughput Tracking of Pluripotent Human Embryonic Stem Cells with Dual Fluorescence Resonance Energy Transfer Molecular Beacons

Pluripotent human embryonic stem cells (hESCs) provide an unprecedented opportunity for the study of human tissue development, and the development of cell-based therapies for human disease. To realize these potential advances, however, methods for monitoring expression of intracellular proteins in live hESCs without altering cellular properties are needed. Molecular beacons are single-stranded oligonucleotides that have been employed to assay gene expression. To test their potential for high-throughput isolation of hESCs, we developed a dual fluorescence resonance energy transfer (FRET) molecular beacon system using fluorescence-activated cell sorting (FACS) with Oct4 as a target. We demonstrate that Oct4 can be detected by FRET using confocal microscopy, that this can be applied in a high-throughput manner to the identification and isolation of Oct4-expressing hESCs by FACS, that FRET-positive hESCs demonstrate pluripotency in culture and in vivo, and that hESCs transfected with molecular beacons demonstrate normal growth rates and oligonucleotide extinction over time. These studies demonstrate that FRET-based FACS using molecular beacons provides a useful tool for isolating Oct4-expressing pluripotent hESCs, and may also be adapted to selecting differentiating hESCs at specific developmental time points determined by transcription factor expression without functional or genomic alteration. As such, it provides an important new method for high-throughput isolation of hESC-derived tissue-specific precursors for analytic and therapeutic purposes

A perplexing case of superficial granulomatous pyoderma with sporotrichoid-like distribution

Superficial granulomatous pyoderma (SGP) is a rare pyoderma gangrenosum (PG) variant that differs from classic PG in that the ulcers tend to be more superficial, lack a rapidly advancing border, and are not typically associated with an underlying systemic disease. The ulcers are most commonly painless and located on the trunk, with a clean granulating base. They generally do not show undermining but may have a vegetative border. Lesions usually respond well to either topical or intralesional corticosteroids with complete healing. The classic histopathologic finding is a "three-layer granuloma" in the superficial dermis consisting of central neutrophilic inflammation and necrosis, a surrounding layer of histiocytes and multinucleated giant cells, and an outer most layer of plasma cells and eosinophils. Herein, we present a unique case of SGP with sporotrichoid-like distribution on the lower extremity