Isolation and purification of self-renewable human neural stem cells for cell therapy in experimental model of ischemic stroke.
Methods in molecular biology (Clifton, N.J.)
2013; 1059: 157-167
Neuroprotective effects and magnetic resonance imaging of mesenchymal stem cells labeled with SPION in a rat model of Huntington's disease
STEM CELL RESEARCH
2012; 9 (2): 143-155
Human embryonic stem cells (hESCs) are pluripotent with a strong self-renewable ability making them a virtually unlimited source of neural cells for structural repair in neurological disorders. Currently, hESCs are one of the most promising cell sources amenable for commercialization of off-shelf cell therapy products. However, along with this strong proliferative capacity of hESCs comes the tumorigenic potential of these cells after transplantation. Thus, the isolation and purification of a homogeneous, population of neural stem cells (hNSCs) are of paramount importance to avoid tumor formation in the host brain. This chapter describes the isolation, neuralization, and long-term perpetuation of hNSCs derived from hESCs through use of specific mitogenic growth factors and the preparation of hNSCs for transplantation in an experimental model of stroke. Additionally, we describe methods to analyze the stroke and size of grafts using magnetic resonance imaging and Osirix software, and neuroanatomical tracing procedures to study axonal remodeling after stroke and cell transplantation.
View details for DOI 10.1007/978-1-62703-574-3_14
View details for PubMedID 23934842
The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2012; 419 (1): 38-42
Bone marrow mesenchymal stem cells (MSC) have been tested and proven effective in some neurodegenerative diseases, but their tracking after transplantation may be challenging. Our group has previously demonstrated the feasibility and biosafety of rat MSC labeling with iron oxide superparamagnetic nanoparticles (SPION). In this study, we investigated the therapeutic potential of SPION-labeled MSC in a rat model of Huntington's disease, a genetic degenerative disease with characteristic deletion of striatal GABAergic neurons. MSC labeled with SPION were injected into the striatum 1h after quinolinic acid injection. FJ-C analysis demonstrated that MSC transplantation significantly decreased the number of degenerating neurons in the damaged striatum 7 days after lesion. In this period, MSC transplantation enhanced the striatal expression of FGF-2 but did not affect subventricular zone proliferation, as demonstrated by Ki67 proliferation assay. In addition, MSC transplantation significantly reduced the ventriculomegaly in the lesioned brain. MRI and histological techniques detected the presence of the SPION-labeled cells at the lesion site. SPION-labeled MSC produced magnetic resonance imaging (MRI) signals that were visible for at least 60 days after transplantation. Our data highlight the potential of adult MSC to reduce brain damage under neurodegenerative diseases and indicate the use of nanoparticles in cell tracking, supporting their potential as valuable tools for cell therapy.
View details for DOI 10.1016/j.scr.2012.05.005
View details for Web of Science ID 000308453100009
View details for PubMedID 22742973
Human menstrual blood-derived mesenchymal cells as a cell source of rapid and efficient nuclear reprogramming.
2012; 21 (10): 2215-2224
The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.
View details for DOI 10.1016/j.bbrc.2012.01.119
View details for Web of Science ID 000301560500007
View details for PubMedID 22326867
Cysteine Proteases in Differentiation of Embryonic Stem Cells into Neural Cells
STEM CELLS AND DEVELOPMENT
2011; 20 (11): 1859-1872
Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes-OCT4, KLF4, SOX2, and c-MYC-in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2, and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days of posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2-5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.
View details for DOI 10.3727/096368912X653048
View details for PubMedID 22776164
Leishmania Inhibitor of Serine Peptidase 2 Prevents TLR4 Activation by Neutrophil Elastase Promoting Parasite Survival in Murine Macrophages
JOURNAL OF IMMUNOLOGY
2011; 186 (1): 411-422
Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of β-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of β-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required β-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.
View details for DOI 10.1089/scd.2010.0186
View details for Web of Science ID 000296587400004
View details for PubMedID 21417836
Isolation of neurosphere-like bodies from an adult patient with refractory temporal lobe epilepsy
ARQUIVOS DE NEURO-PSIQUIATRIA
2010; 68 (6): 956-958
Heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes
2007; 145 (3-4): 234-239
Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to parasite killing. Whereas all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote parasite survival and growth.
View details for DOI 10.4049/jimmunol.1002175
View details for Web of Science ID 000285688700048
View details for PubMedID 21098233
We have examined the heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes (HBP-Lb) by chromatography assays. The proposed strategy to isolate an enriched fraction of the HBP-Lb consisted of an association of the Triton X-114 method with affinity chromatography in heparin-Sepharose 4B column. SDS-PAGE analysis of the eluted proteins showed two main protein bands (65.0 and 54.5 kDa), while a single protein band was observed in native electrophoresis gel. The hemagglutination property of HBP-Lb over rabbit erythrocytes was confirmed up to 6.3+/-0.5 microg of protein mL(-1). Additionally, we have assayed the potential of HBP-Lb labeled with sulfo-NHS-LC-biotin in binding to nitrocellulose-immobilized gut proteins extracted of Lutzomyia intermedia and Lutzomyia whitmani. The results indicated a similar profile of five ligands (67.0, 62.1, 59.5, 56.0 and 47.5 kDa) in both studied Lutzomyia species. This is the first direct description of this class of protein in L. (V.) braziliensis with a suggestion of its biological activity in the interaction of Leishmania with Lutzomyia gut cells, which maybe a crucial step during this parasite's life cycle.
View details for DOI 10.1016/j.vetpar.2006.12.019
View details for Web of Science ID 000245797000005
View details for PubMedID 17292553