Jorge B. Aquino

Schwann Cell Precursors from Nerve Innervation Are a Cellular Origin of Melanocytes in Skin

Current opinion holds that pigment cells, melanocytes, are derived from neural crest cells produced at the dorsal neural tube and that migrate under the epidermis to populate all parts of the skin. Here, we identify growing nerves projecting throughout the body as a stem/progenitor niche containing Schwann cell precursors (SCPs) from which large numbers of skin melanocytes originate. SCPs arise as a result of lack of neuronal specification by Hmx1 homeobox gene function in the neural crest ventral migratory pathway. Schwann cell and melanocyte development share signaling molecules with both the glial and melanocyte cell fates intimately linked to nerve contact and regulated in an opposing manner by Neuregulin and soluble signals including insulin-like growth factor and platelet-derived growth factor. These results reveal SCPs as a cellular origin of melanocytes, and have broad implications on the molecular mechanisms regulating skin pigmentation during development, in health and pigmentation disorders.

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis

Hepatocyte apoptosis, the hallmark of non‐alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine‐proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan‐caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH.

In vitro and in vivo differentiation of boundary cap neural crest stem cells into mature Schwann cells.

Boundary cap cells can generate neurons as well as peripheral glia during embryonic development (Maro, G.S., Vermeren, M., Voiculescu, O., Melton, L., Cohen, J., Charnay, P., Topilko, P., 2004. Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS. Nat Neurosci. 7 (9), 930-938), and, recently, the boundary cap was shown to contain multipotent stem cells (Hjerling-Leffler, J., Marmigère, F., Heglind, M., Cederberg, A., Koltzenburg, M., Enerbäck, S., Ernfors, P., 2005. The boundary cap, a source of neural crest stem cells generating multiple sensory neuron subtypes. Development. 132 (11), 2623-2632). The ability of stem cells to generate mature functional glial phenotypes has not been addressed. In this study, we have explored the competence of boundary neural crest stem cells (bNCSCs) to differentiate into mature functional Schwann cells (SCs) in vitro and in vivo. bNCSCs failed to differentiate into SCs in vitro when cultured in a defined media and in vivo when grafted into adult rat sciatic nerves. However, in the presence of neuregulins, during long-term cultures, the majority of bNCSCs differentiated into SCs. After analysis of the in vivo expression of Sox2, Sox10, S100, GFAP, fibronectin and Krox20 in the glial lineages, we used these markers to characterize differentiation of the bNCSCs. Gliogenesis of bNCSCs proceeded similar to that in vivo by sequentially adopting a SC precursor and immature Schwann cell before maturing into myelinating and non-myelinating SCs. In co-culture with explanted dorsal root ganglia (DRG) as well as in vivo in transplants to the axotomized sciatic nerve, these bNCSC-derived SCs myelinated axons as shown by ensheathing of neuronal processes and expression of myelin basic proteins (MBP). These results show that, under appropriate conditions, bNCSCs can generate mature SCs that are functional and can myelinate axons in regenerating nerves.

Mesenchymal stem cells as therapeutic tools and gene carriers in liver fibrosis and hepatocellular carcinoma

Mesenchymal stem (stromal) cells (MSCs) are a source of circulating progenitors that are able to generate cells of all mesenchymal lineages and to cover cellular demands of injured tissues. The extent of their transdifferentiation plasticity remains controversial. Cells with MSC properties have been obtained from diverse tissues after purification and expansion in vitro. These cellular populations are heterogeneous and under certain conditions show pluripotent-like properties. MSCs present immunosuppressive and anti-inflammatory features and high migratory capacity toward inflamed or remodeling tissues. In this study we review available data regarding factors and signaling axes involved in the chemoattraction and engraftment of MSCs to an injured tissue or to a tissue undergoing active remodeling. Moreover, experimental evidence in support of uses of MSCs as vehicles of therapeutic genes is discussed. Because of its regenerative capacity and its particular immune properties, the liver is a good model to analyze the potential of MSC-based therapies. Finally, the potential application of MSCs and genetically modified MSCs in liver fibrosis and hepatocellular carcinoma (HCC) is proposed in view of available evidence.

Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.

Adenovirus-mediated inhibition of SPARC attenuates liver fibrosis in rats.

The interaction between fibrogenic cells and extracellular matrix plays a role in liver fibrosis, yet the mechanisms are largely unknown. Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is expressed by hepatic stellate cells and is overexpressed in fibrotic livers. We investigated the in vivo role of SPARC in experimentally induced liver fibrosis in rats.

Lack of the Central Nervous System- and Neural Crest-Expressed Forkhead Gene Foxs1 Affects Motor Function and Body Weight

ABSTRACT To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-β-galactosidase reporter gene “knock-in” (Foxs1β-gal/β-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a β-galactosidase reporter gene. Staining for β-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1β-gal/β-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1β-gal/lβ-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1β-gal/β-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.

The retinoic acid inducible Cas-family signaling protein Nedd9 regulates neural crest cell migration by modulating adhesion and actin dynamics.

Cell migration is essential for the development of numerous structures derived from embryonic neural crest cells (NCCs), however the underlying molecular mechanisms are incompletely understood. NCCs migrate long distances in the embryo and contribute to many different cell types, including peripheral neurons, glia and pigment cells. In the present work we report expression of Nedd9, a scaffolding protein within the integrin signaling pathway, in non-lineage-restricted neural crest progenitor cells. In particular, Nedd9 was found to be expressed in the dorsal neural tube at the time of neural crest delamination and in early migrating NCCs. To analyze the role of Nedd9 in neural crest development we performed loss- and gain-of-function experiments and examined the subsequent effects on delamination and migration in vitro and in vivo. Our results demonstrate that loss of Nedd9 activity in chick NCCs perturbs cell spreading and the density of focal complexes and actin filaments, properties known to depend on integrins. Moreover, a siRNA dose-dependent decrease in Nedd9 activity results in a graded reduction of NCCs migratory distance while forced overexpression increases it. Retinoic acid (RA) was found to regulate Nedd9 expression in NCCs. Our results demonstrate in vivo that Nedd9 promotes the migration of NCCs in a graded manner and suggest a role for RA in the control of Nedd9 expression levels.

Hepatocellular carcinoma cells and their fibrotic microenvironment modulate bone marrow-derived mesenchymal stromal cell migration in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death. Fibrogenesis is an active process characterized by the production of several proinflammatory cytokines, chemokines and growth factors. It involves the activation of hepatic stellate cells (HSCs) which accumulate at the site of injury and are the main source of the extracellular matrix deposits. There are no curative treatments for advanced HCC, thus, new therapies are urgently needed. Mesenchymal stromal cells (MSCs) have the ability to migrate to sites of injury or to remodeling tissues after in vivo administration; however, in several cancer models they demonstrated limited efficacy to eradicate experimental tumors partially due to poor engraftment. Thus, the aim of this work was to analyze the capacity of human MSCs (hMSCs) to migrate and anchor to HCC tumors. We observed that HCC and HSCs, but not nontumoral stroma, produce factors that induce hMSC migration in vitro. Conditioned media (CM) generated from established HCC cell lines were found to induce higher levels of hMSC migration than CM derived from fresh patient tumor samples. In addition, after exposure to CM from HCC cells or HSCs, hMSCs demonstrated adhesion and invasion capability to endothelial cells, type IV collagen and fibrinogen. Consistently, these cells were found to increase metalloproteinase-2 activity. In vivo studies with subcutaneous and orthotopic HCC models indicated that intravenously infused hMSCs migrated to lungs, spleen and liver. Seven days post-hMSC infusion cells were located also in the tumor in both models, but the signal intensity was significantly higher in orthotopic than in subcutaneous model. Interestingly, when orthotopic HCC tumors where established in noncirrhotic or cirrhotic livers, the amount of hMSCs localized in the liver was higher in comparison with healthy animals. A very low signal was found in lungs and spleens, indicating that liver tumors are able to recruit them at high efficiency. Taken together our results indicate that HCC and HSC cells produce factors that efficiently induce hMSC migration toward tumor microenvironment in vitro and in vivo and make MSCs candidates for cell-based therapeutic strategies to hepatocellular carcinoma associated with fibrosis.

Low molecular weight hyaluronan inhibits colorectal carcinoma growth by decreasing tumor cell proliferation and stimulating immune response.

Hyaluronan modulates cancer progression by multiple mechanisms; nevertheless, its effects remain controversial. In this work, low molecular weight (LMW) hyaluronan but not high molecular weight (HMW) was found to significantly reduce colorectal carcinoma (CRC) growth in vitro and in vivo. Both survival and proliferation of CT26 tumor cells were affected by treatment with low doses of LMW HA, with involvement of Akt signaling mechanisms. We show for the first time that splenocytes isolated from LMW HA-treated animals present significantly higher proliferative capacity upon stimulation with dendritic cells (DCs) pulsed with tumor lysate. Consistently, expression of MHC class II and costimulatory molecules were increased in DCs isolated from the spleen of LMW HA-treated mice. Besides, increased tumor infiltrating lymphocytes was observed in animals treated with LMW HA. Our results suggest that LMW HA in a model of CRC triggers an activation of immune system, which is likely involved in the observed tumor growth inhibition. LMW HA is suggested as a candidate molecule for therapeutic adjuvant treatments in CRC immunotherapy.