Mario Soriano

Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution

Thymidine analogs, including bromodeoxyuridine, chlorodeoxyuridine, iododeoxyuridine, and tritiated thymidine, label dividing cells by incorporating into DNA during S phase of cell division and are widely employed to identify cells transplanted into the central nervous system. However, the potential for transfer of thymidine analogs from grafted cells to dividing host cells has not been thoroughly tested. We here demonstrate that graft‐derived thymidine analogs can become incorporated into host neural precursors and glia. Large numbers of labeled neurons and glia were found 3–12 weeks after transplantation of thymidine analog‐labeled live stem cells, suggesting differentiation of grafted cells. Remarkably, however, similar results were obtained after transplantation of dead cells or labeled fibroblasts. Our findings reveal for the first time that thymidine analog labeling may not be a reliable means of identifying transplanted cells, particularly in highly proliferative environments such as the developing, neurogenic, or injured brain.

Absence of Dysferlin Alters Myogenin Expression and Delays Human Muscle Differentiation “in Vitro”

Mutations in dysferlin cause a type of muscular dystrophy known as dysferlinopathy. Dysferlin may be involved in muscle repair and differentiation. We compared normal human skeletal muscle cultures expressing dysferlin with muscle cultures from dysferlinopathy patients. We quantified the fusion index of myoblasts as a measure of muscle development and conducted optic and electronic microscopy, immunofluorescence, Western blot, flow cytometry, and real-time PCR at different developmental stages. Short interference RNA was used to corroborate the results obtained in dysferlin-deficient cultures. A luciferase reporter assay was performed to study myogenin activity in dysferlin-deficient cultures. Myoblasts fusion was consistently delayed as compared with controls whereas the proliferation rate did not change. Electron microscopy showed that control cultured cells at 10 days were fusiform, whereas dysferlin-deficient cells were star-shaped and large. After 15 days the normal multinucleated appearance and structured myofibrils were not present in dysferlin-deficient cells. Strikingly, myogenin was not detected in myotubes from dysferlin-deficient cultures using Western blot, and mRNA analysis showed low levels (p < 0.05) compared with controls. Flow cytometry and immunofluorescence also showed reduced levels of myogenin in dysferlin-deficient cultures. When the dysferlin gene was knocked down (∼80%), myogenin mRNA leveled down to ∼70%. MyoD and desmin mRNA levels in controls and dysferlin-deficient cultures were similar. The reporter luciferase assay demonstrated a low myogenin activity in dysferlin-deficient cultures. These results point to a functional link between dysferlin and myogenin, and both proteins may share a new signaling pathway involved in differentiation of skeletal muscle in vitro.

Regulation of neurogenesis by neurotrophins in developing spinal sensory ganglia

Neurons and glia in spinal sensory ganglia derive from multipotent neural crest-derived stem cells. In contrast to neural progenitor cells in the central nervous system, neural crest progenitors coexist with differentiated sensory neurons all throughout the neurogenic period. Thus, developing sensory ganglia are advantageous for determining the possible influence of cell-cell interactions in the regulation of precursor proliferation and neurogenesis. Neurotrophins are important regulators of neuronal survival in the developing vertebrate nervous system and, in addition, they appear to influence precursor behavior in vitro. Studies in mice carrying mutations in neurotrophin genes provide a good system in which to analyze essential actions of these factors on the different developing neural populations.

Epicardial delivery of collagen patches with adipose-derived stem cells in rat and minipig models of chronic myocardial infarction.

Although transplantation of adipose-derived stem cells (ADSC) in chronic myocardial infarction (MI) models is associated with functional improvement, its therapeutic value is limited due to poor long-term cell engraftment and survival. Thus, the objective of this study was to examine whether transplantation of collagen patches seeded with ADSC could enhance cell engraftment and improve cardiac function in models of chronic MI. With that purpose, chronically infarcted Sprague-Dawley rats (n = 58) were divided into four groups and transplanted with media, collagen scaffold (CS), rat ADSC, or CS seeded with rat ADSC (CS-rADSC). Cell engraftment, histological changes, and cardiac function were assessed 4 months after transplantation. In addition, Göttingen minipigs (n = 18) were subjected to MI and then transplanted 2 months later with CS or CS seeded with autologous minipig ADSC (CS-pADSC). Functional and histological assessments were performed 3 months post-transplantation. Transplantation of CS-rADSC was associated with increased cell engraftment, significant improvement in cardiac function, myocardial remodeling, and revascularization. Moreover, transplantation of CS-pADSC in the pre-clinical swine model improved cardiac function and was associated with decreased fibrosis and increased vasculogenesis. In summary, transplantation of CS-ADSC resulted in enhanced cell engraftment and was associated with a significant improvement in cardiac function and myocardial remodeling.

Adipose stromal vascular fraction improves cardiac function in chronic myocardial infarction through differentiation and paracrine activity.

Fresh adipose-derived cells have been shown to be effective in the treatment of acute myocardial infarction (MI), but their role in the chronic setting is unknown. We sought to determine the long-term effect of the adipose derived-stromal vascular fraction (SVF) cell transplantation in a rat model of chronic MI. MI was induced in 82 rats by permanent coronary artery ligation and 5 weeks later rats were allocated to receive an intramyocardial injection of 107 GFP-expressing fresh SVF cells or culture media as control. Heart function and tissue metabolism were determined by echocardiography and 18F-FDG-microPET, respectively, and histological studies were performed for up to 3 months after transplantation. SVF induced a statistically significant long-lasting (3 months) improvement in cardiac function and tissue metabolism that was associated with increased revascularization and positive heart remodeling, with a significantly smaller infarct size, thicker infarct wall, lower scar fibrosis, and lower cardiac hypertrophy. Importantly, injected cells engrafted and were detected in the treated hearts for at least 3 months, directly contributing to the vasculature and myofibroblasts and at negligible levels to cardiomyocytes. Furthermore, SVF release of angiogenic (VEGF and HGF) and proinflammatory (MCP-1) cytokines, as well as TIMP1 and TIMP4, was demonstrated in vitro and in vivo, strongly suggesting that they have a trophic effect. These results show the potential of SVF to contribute to the regeneration of ischemic tissue and to provide a long-term functional benefit in a rat model of chronic MI, by both direct and indirect mechanisms.

Sp1 Transcription Factor Interaction with Accumulated Prelamin A Impairs Adipose Lineage Differentiation in Human Mesenchymal Stem Cells: Essential Role of Sp1 in the Integrity of Lipid Vesicles

Lamin A (LMNA)‐linked lipodystrophies may be either genetic (associated with LMNA mutations) or acquired (associated with the use of human immunodeficiency virus protease inhibitors [PIs]), and in both cases they share clinical features such as anomalous distribution of body fat or generalized loss of adipose tissue, metabolic alterations, and early cardiovascular complications. Both LMNA‐linked lipodystrophies are characterized by the accumulation of the lamin A precursor prelamin A. The pathological mechanism by which prelamin A accumulation induces the lipodystrophy associated phenotypes remains unclear. Since the affected tissues in these disorders are of mesenchymal origin, we have generated an LMNA‐linked experimental model using human mesenchymal stem cells treated with a PI, which recapitulates the phenotypes observed in patient biopsies. This model has been demonstrated to be a useful tool to unravel the pathological mechanism of the LMNA‐linked lipodystrophies, providing an ideal system to identify potential targets to generate new therapies for drug discovery screening. We report for the first time that impaired adipogenesis is a consequence of the interaction between accumulated prelamin A and Sp1 transcription factor, sequestration of which results in altered extracellular matrix gene expression. In fact, our study shows a novel, essential, and finely tuned role for Sp1 in adipose lineage differentiation in human mesenchymal stem cells. These findings define a new physiological experimental model to elucidate the pathological mechanisms LMNA‐linked lipodystrophies, creating new opportunities for research and treatment not only of LMNA‐linked lipodystrophies but also of other adipogenesis‐associated metabolic diseases.

Improved technique for stereotactic placement of nerve grafts between two locations inside the rat brain.

Peripheral nerve grafts have shown the ability to facilitate central axonal growth and regenerate the adult central nervous system. However, the detailed description of a technique for atraumatic graft placement within the brain is lacking. We present a stereotactic procedure to implant a peripheral nerve graft within a rats brain with minimal brain tissue damage. The procedure permits a correct graft placement joining two chosen points, and the survival and integration of the graft in the host tissue with a light glial reaction, with evidence of central axonal growth inside the graft, at least up to 8 weeks after its implantation.

Enteric neurons show a primary cilium

The primary cilium is a non‐motile cilium whose structure is 9+0. It is involved in co‐ordinating cellular signal transduction pathways, developmental processes and tissue homeostasis. Defects in the structure or function of the primary cilium underlie numerous human diseases, collectively termed ciliopathies. The presence of single cilia in the central nervous system (CNS) is well documented, including some choroid plexus cells, neural stem cells, neurons and astrocytes, but the presence of primary cilia in differentiated neurons of the enteric nervous system (ENS) has not yet been described in mammals to the best of our knowledge. The enteric nervous system closely resembles the central nervous system. In fact, the ultrastructure of the ENS is more similar to the CNS ultrastructure than to the rest of the peripheral nervous system. This research work describes for the first time the ultrastructural characteristics of the single cilium in neurons of rat duodenum myenteric plexus, and reviews the cilium function in the CNS to propose the possible role of cilia in the ENS cells.

Confocal technology in fluorescence in situ hybridization evaluation for cancer: a diagnostic improvement.

During the cancer routine diagnosis course, we commonly use fluorescence in situ hybridization (FISH) technique. FISH studies are conducted for genes amplification analyses (ErBb2/Neu) and also for genes translocation studies such as CMYC, BCL6, or BCL2. Usually, FISH evaluation is carried out with fluorescence microscopy and photographed with sensitive cameras. An alternative technology to the fluorescence microscopy is using the confocal microscopy for the evaluation of these samples. Some advantages of confocal microscopy are as follows: First, the use of a laser and pinhole instead of using 511983 IJSXXX10.1177/1066896913511983International Journal of Surgical PathologyCampos et al. research-article2013