Human Myoblast and Mesenchymal Stem Cell Interactions Visualized by Videomicroscopy.

Muscle-derived progenitor cell (myoblast) therapy has promise for the treatment of denervated, weakened, and fibrotic muscle. The best methods for injecting myoblasts to promote fusion and retention have yet to be determined, however. Mesenchymal stem/stromal cells have also been reported to have beneficial effects in restoring damaged tissue, through increasing vascularization and reducing inflammation. The interactions between human primary skeletal myoblasts and bone marrow-derived mesenchymal stem/stromal cells were examined using time-lapse images put into video format. Of interest, there is a high degree of cell-to-cell interaction with microparticles transferring between both cell types, and formation of nanotubules to bridge cytoplasmic contents between the two types of cell. This model provides an in vitro platform for examining mechanisms for cell-to-cell interaction preceding myoblast fusion.

Intravitreal autologous bone marrow CD34+ cell therapy for ischemic and degenerative retinal disorders: preliminary phase 1 clinical trial findings.

PURPOSE: Because human bone marrow (BM) CD34+ stem cells home into damaged tissue and may play an important role in tissue repair, this pilot clinical trial explored the safety and feasibility of intravitreal autologous CD34+ BM cells as potential therapy for ischemic or degenerative retinal conditions. METHODS: This prospective study enrolled six subjects (six eyes) with irreversible vision loss from retinal vascular occlusion, hereditary or nonexudative age-related macular degeneration, or retinitis pigmentosa. CD34+ cells were isolated under Good Manufacturing Practice conditions from the mononuclear cellular fraction of the BM aspirate using a CliniMACs magnetic cell sorter. After intravitreal CD34+ cell injection, serial ophthalmic examinations, microperimetry/perimetry, fluorescein angiography, electroretinography (ERG), optical coherence tomography (OCT), and adaptive optics OCT were performed during the 6-month follow-up. RESULTS: A mean of 3.4 million (range, 1-7 million) CD34+ cells were isolated and injected per eye. The therapy was well tolerated with no intraocular inflammation or hyperproliferation. Best-corrected visual acuity and full-field ERG showed no worsening after 6 months. Clinical examination also showed no worsening during follow-up except among age-related macular degeneration subjects in whom mild progression of geographic atrophy was noted in both the study eye and contralateral eye at 6-month follow-up, concurrent with some possible decline on multifocal ERG and microperimetry. Cellular in vivo imaging using adaptive optics OCT showed changes suggestive of new cellular incorporation into the macula of the hereditary macular degeneration study eye. CONCLUSIONS: Intravitreal autologous BM CD34+ cell therapy appears feasible and well tolerated in eyes with ischemic or degenerative retinal conditions and merits further exploration. (ClinicalTrials.gov number, NCT01736059.).

Mesenchymal stem cells for the sustained in vivo delivery of bioactive factors.

Mesenchymal stem cells (MSC) are a promising tool for cell therapy, either through direct contribution to the repair of bone, tendon and cartilage or as an adjunct therapy through protein production and immune mediation. They are an attractive vehicle for cellular therapies due to a variety of cell intrinsic and environmentally responsive properties. Following transplantation, MSC are capable of systemic migration, are not prone to tumor formation, and appear to tolerize the immune response across donor mismatch. These attributes combine to allow MSC to reside in many different tissue types without disrupting the local microenvironment and, in some cases, responding to the local environment with appropriate protein secretion. We describe work done by our group and others in using human MSC for the sustained in vivo production of supraphysiological levels of cytokines for the support of cotransplanted hematopoietic stem cells and enzymes that are deficient in animal models of lysosomal storage disorders such as MPSVII. In addition, the use of MSC engineered to secrete protein products has been reviewed in several fields of tissue injury repair, including but not limited to revascularization after myocardial infarction, regeneration of intervertebral disc defects and spine therapy, repair of stroke, therapy for epilepsy, skeletal tissue repair, chondrogenesis/knee and joint repair, and neurodegenerative diseases. Genetically engineered MSC have thus proven safe and efficacious in numerous animal models of disease modification and tissue repair and are poised to be tested in human clinical trials. The potential for these interesting cells to secrete endogenous or transgene products in a sustained and long-term manner is highly promising and is discussed in the current review.

Mutation of critical serine residues in HIV-1 matrix result in an envelope incorporation defect which can be rescued by truncation of the gp41 cytoplasmic tail.

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) domain is involved in both early and late events of the viral life cycle. Simultaneous mutation of critical serine residues in MA has been shown previously to dramatically reduce phosphorylation of MA. However, the role of phosphorylation in viral replication remains unclear. Viruses harboring serine to alanine substitutions at positions 9, 67, 72, and 77 are severely impaired in their ability to infect target cells. In addition, the serine mutant viruses are defective in their ability to fuse with target cell membranes. Interestingly, both the fusion defect and the infectivity defect can be rescued by truncation of the long cytoplasmic tail of gp41 envelope protein (gp41CT). Sucrose density gradient analysis also reveals that these mutant viruses have reduced levels of gp120 envelope protein incorporated into the virions as compared to wild type virus. Truncation of the gp41CT rescues the envelope incorporation defect. Here we propose a model in which mutation of specific serine residues prevents MA interaction with lipid rafts during HIV-1 assembly and thereby impairs recruitment of envelope to the sites of viral budding.

Antiviral activity of a Rac GEF inhibitor characterized with a sensitive HIV/SIV fusion assay.

A virus-dependent fusion assay was utilized to examine the activity of a panel of HIV-1, -2, and SIV isolates of distinct coreceptor phenotypes. This assay allowed identification of entry inhibitors, and characterization of an antagonist of a Rac guanine nucleotide exchange factor, as an inhibitor of HIV-mediated fusion.

IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent.

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E2 (PGE2) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.