Bilateral Crystalline Corneal Deposits as First Clinical Manifestation of Monoclonal Gammopathy: A Case Report

Aims: To report the clinical and diagnostic findings of a patient with bilateral corneal deposits caused by an underlying monoclonal gammopathy. Methods: Slit-lamp biomicroscopy, confocal microscopy and additional serological tests were performed on a 35-year-old man presenting with bilateral crystalline corneal deposits. Results: The patient was diagnosed as having monoclonal gammopathy based on elevated levels of serum immunoglobulin G. Confocal microscopy showed highly reflective (protein) deposits throughout the entire cornea, with the highest density in the epithelium and anterior stromal keratocytes. Conclusions: Monoclonal gammopathy, a potential sign of a life-threatening disease, can lead to dense, bilateral corneal deposits. As such changes can occur long before ocular or systemic discomforts appear, an early diagnosis is crucial. Ophthalmologists should be aware of corneal deposits as potential warning signs of monoclonal gammopathy

Microcarrier-Based Expansion of Adult Murine Side Population Stem Cells

The lack of reliable methods to efficiently isolate and propagate stem cell populations is a significant obstacle to the advancement of cell-based therapies for human diseases. One isolation technique is based on efflux of the fluorophore Hoechst 33342. Using fluorescence-activated cell sorting (FACS), a sub-population containing adult stem cells has been identified in a multitude of tissues in every mammalian species examined. These rare cells are referred to as the ‘side population’ or SP due to a distinctive FACS profile that results from weak staining by Hoechst dye. Although the SP contains multi-potent cells capable of differentiating toward hematopoietic and mesenchymal lineages; there is currently no method to efficiently expand them. Here, we describe a spinner-flask culture system containing C2C12 myoblasts attached to spherical microcarriers that act to support the growth of non-adherent, post-natal murine skeletal muscle and bone marrow SP cells. Using FACS and hemocytometry, we show expansion of unfractionated EGFP+ SP cells over 6 wks. A significant number of these cells retain characteristics of freshly-isolated, unfractionated SP cells with respect to protein expression and dye efflux capacity. Expansion of the SP will permit further study of these heterogeneous cells and determine their therapeutic potential for regenerative and reparative therapies

Appearance of microcarrier-based cultures from stirred-flasks.

<p>A – A schematic representation of the initial steps in establishing a SP expansion culture. ♀ C2C12 myoblasts are expanded and seeded on 500,000 Cytodex 1 microcarriers (left). After attachment to the microcarrier surface, cultures are transferred to 100 mL flasks and maintained for 2–3 days. Freshly-isolated bone marrow (BM) or skeletal muscle (SkM) SP cells isolated from ♂ EGFP-expressing mice are added and stirred at 30 rpm. B – Fluorescent staining of C2C12-containing Cytodex 1 microcarriers using DAPI (blue), an α-sarcomeric actin antibody detected with a conjugated secondary antibody (green), and Texas Red-X phalloidin (red). The combined image is on the right and the scale bar represents 100 µm. C – A closer view of a cellularized microcarrier stained for F-actin and DNA (left) as well as 2 scanning electron micrographs (SEMs). The SEMs were pseudo-colored to emphasize the dextran matrix on the microcarrier surface. Scale bars equal 100 µm (left and center) and 50 µm (right). D – Differential interference contrast, fluorescence, and combined images of day 18 co-cultures of SP cells from bone marrow and myoblast feeder cells. Scale bar equals 100 µm.</p

Phenotypic characterization of stirred-flask expansion cultures.

<p>A – Representative FACS plots of P0 skeletal muscle expansion cultures. The increase in the typical SP gate position in day 15 cultures is shown (left). The plot shows 50,000 events. By day 10, the appearance of a highly-fluorescent (EGFP^high) sub-population was apparent in EGFP⁺ cells and these were found to sort nearly exclusively to the SP gate (right). The EGFP^low cells were distributed in typical SP and MP positions. B – Representative FACS analyses of surface marker expression in P2 bone marrow and P3 skeletal muscle cultures. Plots show 20,000 events. CD45 (left) and Sca-1 (right) profiles plotted against GFP fluorescence are shown for unfractionated aliquots of the suspension cultures. C – Representative immuno-staining results from Cytospin preparations for the EGFP^high (top) and EGFP^low (bottom) sub-populations from skeletal muscle. The EGFP^high images show separate channels depicting DNA staining, GFP fluorescence, ABCG2 immuno-staining, and a combination of fluorescent channels (left to right). EGFP^low images represent the same channels except the red channel shows immuno-staining with MyoD, Pax7, desmin, and ABCG2 (left to right). Scale bars equal 25 µm. D – A summary of immuno-staining experiments for ABCG2, desmin, Pax7, MyoD, myogenin, and Sca-1 in EGFP^high and EGFP^low sub-populations. Percentage values are expressed as Mean ± SD (<i>n</i> = 6). E – A graph showing the increase in the EGFP^high sub-population in skeletal muscle and bone marrow cultures at the end of P0, P1, and P2. The percentage of EGFP^high cells in the EGFP⁺ fraction is expressed as Mean ± SD (<i>n</i> = 5) and asterisks represent statistical significance (* P<0.05 and ** P<0.01) compared to day 15 P0 values.</p

Expansion of bone marrow and skeletal muscle SP cells in suspension.

<p>A – Representative FACS plots of P2 bone marrow suspension culture analyses for EGFP-fluorescence at days 3, 10, and 15 (left). Plots show 20,000 events. Day 15 cells were assessed for Hoechst dye effusion (right). Plots show 50,000 events. B – FACS plots of P2 skeletal muscle culture analyses for EGFP-fluorescence at days 3, 10, and 15 (left). Day 15 cells were assessed for Hoechst dye effusion (right). Both bone marrow and skeletal muscle expansion cultures demonstrated an increase in EGFP⁺ cells over time and many cells were Hoechst^low, analogous to freshly-isolated SP cells. C – Expansion of EGFP⁺ cells in P0 stirred-flask cultures as determined by hemocytometry. Numerical values for the total number of EGFP⁺ cells are expressed as Mean ± SD (<i>n</i> = 4) and asterisks represent statistical significance (* P<0.05 and ** P<0.01) compared to day 3 values. D – Expansion of bone marrow (left) and skeletal muscle (right) EGFP⁺ cells over the course of 3 passages (P0, P1, and P2). The percentage of EGFP⁺ cells in the suspension cultures is expressed as Mean ± SD (<i>n</i> = 4) and asterisks represent statistical significance (* P<0.05 and ** P<0.01) compared to day 3 values.</p

Isolation of SP cells from murine bone marrow and skeletal muscle.

<p>A – Representative FACS plots of bone marrow cells stained with Hoechst 33342 and PI. All plots show 20,000 events. Addition of verapamil eliminated the SP. The linear X and Y axes show PI and Hoechst staining, respectively. B – Representative FACS plots of skeletal muscle cells stained with Hoechst 33342 and PI (20,000 events per plot) in the absence (left) or presence (right) of verapamil. C – The appearance of bone marrow (left) and skeletal muscle (right) SP cells under combined bright field and fluorescence illumination. EGFP-expressing SP cells are shown on a hemocytometer grid and scale bars represent 50 µm (left) and 10 µm (right). D – Representative intrinsic fluorescence in C2C12 cells and unfractionated bone marrow from EGFP-expressing mice. The X axis depicts forward scatter (FSC-H) using a linear scale and the Y axis shows fluorescence using a logarithmic scale. E – Representative fluorescence in MP and SP fractions in enzymatically-digested skeletal muscle from ACTB-EGFP transgenic mice.</p

Cardiac Conduction through Engineered Tissue

In children, interruption of cardiac atrioventricular (AV) electrical conduction can result from congenital defects, surgical interventions, and maternal autoimmune diseases during pregnancy. Complete AV conduction block is typically treated by implanting an electronic pacemaker device, although long-term pacing therapy in pediatric patients has significant complications. As a first step toward developing a substitute treatment, we implanted engineered tissue constructs in rat hearts to create an alternative AV conduction pathway. We found that skeletal muscle-derived cells in the constructs exhibited sustained electrical coupling through persistent expression and function of gap junction proteins. Using fluorescence in situ hybridization and polymerase chain reaction analyses, myogenic cells in the constructs were shown to survive in the AV groove of implanted hearts for the duration of the animal’s natural life. Perfusion of hearts with fluorescently labeled lectin demonstrated that implanted tissues became vascularized and immunostaining verified the presence of proteins important in electromechanical integration of myogenic cells with surrounding recipient rat cardiomyocytes. Finally, using optical mapping and electrophysiological analyses, we provide evidence of permanent AV conduction through the implant in one-third of recipient animals. Our experiments provide a proof-of-principle that engineered tissue constructs can function as an electrical conduit and, ultimately, may offer a substitute treatment to conventional pacing therapy