Albert Edge

Deglycosylation of glycoproteins by trifluoromethanesulfonic acid

Abstract A new method for the chemical deglycosylation of glycoproteins is described. Treatment of fetuin with trifluoromethanesulfonic acid at 0 or 25°C results in rapid cleavage of peripheral sugars, slow loss of serine- and threonine-linked N-acetylgalactosamine, and retention of N-glycosidically linked N-acetylglucosamine. Amino acid analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal that the peptide backbone is left intact after reaction at 0°C for 2.5 h or at 25°C for 1 h. Treatment of ovine luteinizing hormone and human M,N-active erythrocyte sialoglycoproteins at 0°C for 3 h gives similar results. In the case of ovine luteinizing hormone, the recovered deglycosylated hormone has approximately 2% of the starting materials activity as measured in a receptor binding assay. In the case of bovine nasal septum proteoglycan, the deglycosylated core protein acts as an excellent acceptor for UDP- d -xylose:core protein β-O-xylosyltransferase. The reagent is far less toxic than HF and does not require special equipment for handling. The procedure should prove useful not only for generation of deglycosylated proteins, but also for assessment of the number of asparagine-linked saccharide chains in a glycoprotein.

Autologous Skeletal Myoblasts Transplanted to Ischemia-Damaged Myocardium in Humans Histological Analysis of Cell Survival and Differentiation

OBJECTIVES We report histological analysis of hearts from patients with end-stage heart disease who were transplanted with autologous skeletal myoblasts concurrent with left ventricular assist device (LVAD) implantation. BACKGROUND Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. To date, there is only indirect evidence to suggest survival of skeletal muscle in humans. METHODS Five patients (all male; median age 60 years) with ischemic cardiomyopathy, refractory heart failure, and listed for heart transplantation underwent muscle biopsy from the quadriceps muscle. The muscle specimen was shipped to a cell isolation facility where myoblasts were isolated and grown. Patients received a transplant of 300 million cells concomitant with LVAD implantation. Four patients underwent LVAD explant after 68, 91, 141, and 191 days of LVAD support (three transplant, one LVAD death), respectively. One patient remains alive on LVAD support awaiting heart transplantation. RESULTS Skeletal muscle cell survival and differentiation into mature myofibers were directly demonstrated in scarred myocardium from three of the four explanted hearts using an antibody against skeletal muscle-specific myosin heavy chain. An increase in small vessel formation was observed in one of three patients at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells. CONCLUSIONS These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.

Cell Therapy Attenuates Deleterious Ventricular Remodeling and Improves Cardiac Performance After Myocardial Infarction

BackgroundMyocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. Methods and ResultsExperimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 106 myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of ≈30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. ConclusionsImplanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

Notch Inhibition Induces Cochlear Hair Cell Regeneration and Recovery of Hearing after Acoustic Trauma

Hearing loss due to damage to auditory hair cells is normally irreversible because mammalian hair cells do not regenerate. Here, we show that new hair cells can be induced and can cause partial recovery of hearing in ears damaged by noise trauma, when Notch signaling is inhibited by a γ-secretase inhibitor selected for potency in stimulating hair cell differentiation from inner ear stem cells in vitro. Hair cell generation resulted from an increase in the level of bHLH transcription factor Atoh1 in response to inhibition of Notch signaling. In vivo prospective labeling of Sox2-expressing cells with a Cre-lox system unambiguously demonstrated that hair cell generation resulted from transdifferentiation of supporting cells. Manipulating cell fate of cochlear sensory cells in vivo by pharmacological inhibition of Notch signaling is thus a potential therapeutic approach to the treatment of deafness.

Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors in the cochlea.

Auditory hair cells are surrounded on their basolateral aspects by supporting cells, and these two cell types together constitute the sensory epithelium of the organ of Corti, which is the hearing apparatus of the ear. We show here that Lgr5, a marker for adult stem cells, was expressed in a subset of supporting cells in the newborn and adult murine cochlea. Lgr5-expressing supporting cells, sorted by flow cytometry and cultured in a single-cell suspension, compared with unsorted cells, displayed an enhanced capacity for self-renewing neurosphere formation in response to Wnt and were converted to hair cells at a higher (>10-fold) rate. The greater differentiation of hair cells in the neurosphere assay showed that Lgr5-positive cells had the capacity to act as cochlear progenitor cells, and lineage tracing confirmed that Lgr5-expressing cells accounted for the cells that formed neurospheres and differentiated to hair cells. The responsiveness to Wnt of cells with a capacity for division and sensory cell formation suggests a potential route to new hair cell generation in the adult cochlea.

Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function.

The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation, gives rise to a broad range of molecules that can have an identical underlying protein core. An understanding of glycosylation of proteins is important in clarifying the nature of the numerous variants observed and in determining the biological roles of these modifications. Deglycosylation with TFMS (trifluoromethanesulphonic acid) [Edge, Faltynek, Hof, Reichert, and Weber, (1981) Anal. Biochem. 118, 131-137] has been used extensively to remove carbohydrate from glycoproteins, while leaving the protein backbone intact. Glycosylated proteins from animals, plants, fungi and bacteria have been deglycosylated with TFMS, and the most extensively studied types of carbohydrate chains in mammals, the N-linked, O-linked and glycosaminoglycan chains, are all removed by this procedure. The method is based on the finding that linkages between sugars are sensitive to cleavage by TFMS, whereas the peptide bond is stable and is not broken, even with prolonged deglycosylation. The relative susceptibility of individual sugars in glycosidic linkage varies with the substituents at C-2 and the occurrence of amido and acetyl groups, but even the most stable sugars are removed under conditions that are sufficiently mild to prevent scission of peptide bonds. The post-translational modifications of proteins have been shown to be required for diverse biological functions, and selective procedures to remove these modifications play an important role in the elucidation of protein structure and function.

Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells

Stem cells have been demonstrated in the inner ear but they do not spontaneously divide to replace damaged sensory cells. Mesenchymal stem cells (MSC) from bone marrow have been reported to differentiate into multiple lineages including neurons, and we therefore asked whether MSCs could generate sensory cells. Overexpression of the prosensory transcription factor, Math1, in sensory epithelial precursor cells induced expression of myosin VIIa, espin, Brn3c, p27Kip, and jagged2, indicating differentiation to inner ear sensory cells. Some of the cells displayed F-actin positive protrusions in the morphology characteristic of hair cell stereociliary bundles. Hair cell markers were also induced by culture of mouse MSC-derived cells in contact with embryonic chick inner ear cells, and this induction was not due to a cell fusion event, because the chick hair cells could be identified with a chick-specific antibody and chick and mouse antigens were never found in the same cell.

BMP4 induction of sensory neurons from human embryonic stem cells and reinnervation of sensory epithelium

In mammals, hair cells and auditory neurons lack the capacity to regenerate, and damage to either cell type can result in hearing loss. Replacement cells for regeneration could potentially be made by directed differentiation of human embryonic stem (hES) cells. To generate sensory neurons from hES cells, neural progenitors were first made by suspension culture of hES cells in a defined medium. The cells were positive for nestin, a neural progenitor marker, and Pax2, a marker for cranial placodes, and were negative for α‐fetoprotein, an endoderm marker. The precursor cells could be expanded in vitro in fibroblast growth factor (FGF)‐2. Neurons and glial cells were found after differentiation of the neural progenitors by removal of FGF‐2, but evaluation of neuronal markers indicated insignificant production of sensory neurons. Addition of bone morphogenetic protein 4 (BMP4) to neural progenitors upon removal of FGF‐2, however, induced significant numbers of neurons that were positive for markers associated with cranial placodes and neural crest, the sources of sensory neurons in the embryo. Neuronal processes from hES cell‐derived neurons made contacts with hair cells in denervated ex vivo sensory epithelia and expressed synaptic markers, suggesting the formation of synapses. In a gerbil model with a denervated cochlea, the ES cell‐derived neurons engrafted in the auditory nerve trunk and sent out neurites that grew toward the auditory sensory epithelium. These data indicate that hES cells can be induced to form sensory neurons that have the potential to treat neural degeneration associated with sensorineural hearing loss.

Xenogeneic cell therapy: current progress and future developments in porcine cell transplantation.

The multitude of distinct cell types present in mature and developing tissues display unique physiologic characteristics. Cellular therapy is a novel technology with the promise of utilizing this diversity to treat a wide range of human degenerative diseases. Intractable diseases, disorders, and injuries are characterized by cell death or aberrant cellular function. Cell transplantation can replace diseased or lost tissue to provide restorative therapy for these conditions. The limited use of cell transplants as a basis for current therapy can, in part, be attributed to the lack of available human cells suitable for transplantation. This has prevented further realization of the promise of cell transplantation as a platform technology. Accordingly, cell-based therapies such as blood transfusions, for which the cells are readily available, are a standard part of current medical practice. Despite numerous attempts to expand primary human cells in tissue culture, current technological limitations of this approach in regard to proliferative capacity and maintenance of the differentiated phenotype has prevented their use for transplantation. Further, use of human stem cells for the derivation of specific cell types for transplantation is an area of future application with great potential, but hurdles remain in regard to deriving and sufficiently expanding these multipotential cells. Thus, it appears that primary cells are at present a superior source for transplantation. This review focuses on pigs as a source of a variety of primary cells to advance cell therapy to the clinic and implement achievement of its full potential. We outline the advantages and disadvantages of xenogeneic cell therapy while underscoring the utility of transplantable porcine cells for the treatment of human disease.