Cell Animal Models Research Articles

Peripheral Nerve Repair with Cultured Schwann Cells: Getting Closer to the Clinics

Peripheral nerve injuries are a frequent and disabling condition, which affects 13 to 23 per 100.000 persons each year. Severe cases, with structural disruption of the nerve, are associated with poor functional recovery. The experimental treatment using nerve grafts to replace damaged or shortened axons is limited by technical difficulties, invasiveness, and mediocre results. Other therapeutic choices include the adjunctive application of cultured Schwann cells and nerve conduits to guide axonal growth. The bone marrow is a rich source of mesenchymal cells, which can be differentiated in vitro into Schwann cells and subsequently engrafted into the damaged nerve. Alternatively, undifferentiated bone marrow mesenchymal cells can be associated with nerve conduits and afterward transplanted. Experimental studies provide evidence of functional, histological, and electromyographical improvement following transplantation of bone-marrow-derived cells in animal models of peripheral nerve injury. This paper focuses on this new therapeutic approach highlighting its direct translational and clinical utility in promoting regeneration of not only acute but perhaps also chronic cases of peripheral nerve damage.

Ex Vivo Generated Red Cells as Transfusion Products

Ex Vivo Generated Red Cells as Transfusion Products

TCR Gene Editing Results in Effective Immunotherapy of Leukemia without the Development of GvHD

Abstract 667▪▪This icon denotes a clinically relevant abstractTransfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal CD8+ T cells is an attractive strategy for targeted cancer immunotherapy. However, the successful implementation of this approach is limited by technical and safety issues, including inefficient gene transfer, unstable transgene expression, exhaustion of gene-modified cells and most importantly, the unpredictable results of mispairing between the endogenous and exogenous TCR chains. Indeed, co-expression of the endogenous and exogenous TCR in the same cell not only reduces cell-surface expression of the introduced tumor-specific TCR, but also drives the potential for these gene-modified T cells to acquire autoreactive specificities. Such TCR mispairing has been shown to result in autoreactive T cells in animal models [Bendle et al., Nat Med. 2010]. To partially overcome these limitations, we have developed a novel strategy based on zinc finger nucleases (ZFNs) that permits editing of T cell specificity at the DNA level, combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. Two sets of ZFNs were designed targeting the constant regions of the α (TRAC-ZFN) and β (TRBC-ZFN) TCR chain genes, respectively. We transiently delivered these ZFNs into primary T lymphocytes activated with anti-CD3 and anti-CD28 antibody-conjugated beads and cultured with low doses of IL-7 and IL-15, to promote the survival and expansion of the ZFN-modified cells. ZFN delivery into activated T lymphocytes abrogated expression of the CD3/TCR complex on the cell surface (% of CD3neg cells with TRAC-ZFN: 34%±11 and with TRBC-ZFN: 30%±9). No phenotypic differences were observed in CD3pos and CD3neg lymphocytes, which displayed a similar CD4/CD8 ratio while displaying an early T-cell differentiation phenotype, as evidenced by high expression of CD62L, CD27, CD28 and IL-7Rα markers. Sorted CD3neg cells proved stable in culture (demonstrating that ZFN exposure was well tolerated), and did not respond to TCR-dependent stimulation with the mitogen PHA, as expected for cells carrying a disrupted TCR gene. CD3neg cells were efficiently transduced with a lentiviral vector encoding a tumor-specific exogenous TCR chain, resulting in the restoration of cell surface translocation of CD3, thus facilitating the selective expansion of TCR-transduced cells by polyclonal stimulation. To demonstrate the antitumor activity of these modified cells we selected an HLA-A2 restricted, codon-optimized cysteine-modified TCR specific for the Wilms’ tumor antigen 1 (WT1). To achieve complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR α and β chains with high efficiency (averages: 36% and 18%), followed by lentiviral transfer of the tumor-specific TCR α and β chains (average efficiencies: 65% and 25%). This procedure resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition from the endogenous receptor, was expressed at high physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous TCR reactivity including alloreactivity. Finally, in a humanized GvHD model, we showed that, at a variance with 100% of mice infused with unmanipulated T cells, and 80% of mice receiving TCR-transferred lymphocytes developing lethal GvHD, no GvHD was observed upon infusion of matched TCR-edited cells, despite robust T cell engraftment rates across all groups. These data demonstrate that the successful genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific killing activity and thus has the potential to greatly improve the safety and therapeutic benefit of cancer immunotherapy, without triggering its potentially negative effects. (Provasi and Genovese: equal contribution). Disclosures:Liu:Sangamo Biosciences: Employment. Reik:Sangamo Biosciences: Employment. Chu:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Zhang:Sangamo Biosciences: Employment. Bordignon:Molmed: Employment. Holmes:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Bonini:Molmed: Consultancy.

Azacitidine Induces Expansion of Regulatory T Cells and Tumour Specific CD8+ T Lymphocytes After Allogeneic Stem Cell Transplantation: A Strategy for Epigenetic Manipulation of a Graft-Versus-Leukemia Response

Abstract 324Disease relapse and graft-versus-host disease (GVHD) remain the commonest causes of treatment failure after allogeneic stem cell transplantation (SCT) for Acute Myeloid Leukemia (AML). Azacitidine (AZA) possesses inherent anti-leukemic activity but also expands immunomodulatory T regulatory cells in animal models and up-regulates the expression of tumor antigens in vitro. Reasoning that AZA might selectively augment a graft-versus-leukemia (GVL) without a concomitant increase in GVHD we have studied the tolerability and immunological sequelae of AZA administration in patients who have undergone a reduced intensity allogeneic SCT for AML. All patients were transplanted using a conditioning regimen consisting of fludarabine (30 mg/m2 IV × 5 days), melphalan (140 mg/m2IV), and alemtuzumab (10 mg IV × 5 days) with cyclosporine GVHD prophylaxis. Patients received AZA (36 mg/m2) × 5 days every 28 days commencing after sustained neutrophil and platelet engraftment at day +42 with the aim of administering monthly cycles of AZA until 12 months post-transplant. Numbers of CD4+CD25+CD127loFoxP3+ T regulatory cells were measured by flow cytometry. Tumor specific cytotoxic T lymphocytes (CTL) recognising members of the cancer testis antigen (CTA) family and WT1 were quantitated using a CD137 expression and enrichment assay. We report results on 27 patients (median age 59 years) who commenced treatment with AZA (follow-up 3–21 months). 11 patients were transplanted from an HLA identical related donor and 16 from a volunteer unrelated donor. Disease status at the time of transplant was: CR1 n=18; CR2 n=7; first relapse n=2. Post-transplant AZA was well tolerated and 24 patients tolerated at least three cycles of AZA. Haematological toxicity was modest with only two patients experiencing treatment delay for neutropenia, or thrombocytopenia. Three patients developed Grade 2 acute GVHD and no patient developed >Grade 2 acute GVHD. Two patients developed limited chronic GVHD. To date seven patients have relapsed at a median time of 6 months (4–15 months) post-transplant. Administration of AZA had no impact on absolute lymphocyte counts or CD8+ and CD4+ cell numbers compared with a control population of 17 patients transplanted using an identical conditioning regimen. However AZA administration was noted to increase the number of CD4+CD25+CD127loFoxP3+ regulatory T cells within the first 3 months post transplant compared with a time-matched control population (p=0.017) although there was no difference detectable at 6 or 12 months. AZA administration also induced a cytotoxic CD8+ T cell response to candidate tumor antigens MAGE, GAGE, RAGE and WT-1 in the peripheral blood. A CD8+ T cell response to these candidate tumor antigens was only detectable in 1/22 patients pre-transplant but circulating CTA- or WT1 specific CD8+ T cells were detected in 14/16 patients who had received at least six cycles of AZA with a frequency between 0.001–1.4% (mean 0.25%). In four patients with paired peripheral blood and bone marrow samples the size of the CD8+ T cell response was noted to be up to 100 fold greater in the bone marrow. Ex vivo characterisation of the CTA response using dextramers demonstrated the CD8+ T cell response to be effector memory (CD45RA-CCR7-) and functional assays confirmed by mobilization of CD107a and secretion of interferon-g, TNF-a and IL-2 in response to peptide. These data confirm the tolerability of adjunctive AZA post-transplant and its administration appears to be associated with a low incidence of both acute and chronic GVHD. The demonstration that AZA induces both early expansion of T regulatory cells and a tumor specific CD8+ CTL response highlight its potential use as a strategy to epigenetically manipulate a graft-versus-leukemia reaction after allogeneic SCT. Disclosures:Dennis:Celgene: Honoraria, Research Funding. Craddock:Celgene: Honoraria, Research Funding.

Gene Transfer to Hematopoietic Stem/Progenitor Cells As a Novel Approach for Immunotherapy Against B-Lineage Malignancies: In Vivo Xenograft Model,

Abstract 4168Chimeric Antigen Receptors (CAR) against CD19 have been shown to direct T cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. But, there has been insufficient persistence of effector cells, limiting the clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to ensure persistent production of effector cells targeting B-lineage malignant cells, exponentially increasing the number of effectors that may be generated against tumor cells. Experiments were performed using NOD-SCID-IL2 receptor gamma chain null (NSG) mice engrafted with human CD34+ HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. There was efficient and stable transduction with 1–2 copies of CAR/cell as determined by qPCR. Differentiation of modified HSPC in vivo was not impaired by gene transfer, as observed in vitro. Results of in vivo studies showed that CAR-transduced human HSPC successfully differentiated into all lineages, with CAR-expressing T, NK and myeloid cells populating bone marrow, spleen and peripheral blood. The human CD19+ B cell populations normally formed in the xenografted NSG mice were significantly reduced when the transplanted HSPC were transduced with the anti-CD19 CAR, demonstrating in vivo biological activity. Cells harvested from bone marrow and spleen of mice engrafted with modified HSPC lysed CD19-positive cell targets ex vivo. Leukemic challenges of engrafted mice are in progress. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a protocol for generation of effector cells for immunotherapy against B-lineage malignancies. Disclosures:No relevant conflicts of interest to declare.

Abstract C222: Diverting Hsp70 trafficking as a tool for stimulating anticancer immunity.

Abstract The stress-inducible heat shock protein 70 (Hsp70) is normally destined for cytosolic localization. In tumors, Hsp70 is frequently localized to the cell surface and released from cells, unusual features that stimulate the immune system against cancer cells. Interestingly, it has been shown that overexpression of Hsp70 enhances the immunogenicity of tumor cells in animal models. Here we investigated potential mechanisms by which overexpression of Hsp70 in B16 melanoma cells may increase surface expression and release. Flow cytometry measurements revealed an increased cell membrane concentration upon elevated level of intracellular Hsp70. Hsp70 was detectable in subcellular fractions enriched in endosomes/ lysosomes. An accumulation of excess Hsp70 was seen in high Hsp70 expressing cells, in particular in the purified lyosomal fractions. In low expressing cells apparent trafficking of Hsp70 was confined to the endosomal system. In contrast, high intracellular Hsp70 concentration was associated with a predominant lysosomal routing. Remarkable changes were observed in Hsp70 trafficking upon heat stress, a common approach used to facilitate Hsp70 release. In control cells a dramatic depletion of surface-exposed Hsp70 was paralleled with increased endosomal recycling of internalized protein. In contrast, excess intracellular Hsp70 prevented the membrane transport from heat-induced depletion, and allowed transport of surface-exposed Hsp70 to lysosomes secreting their cargo in a soluble form. Therefore excess Hsp70 appears to be decisive in terms of both trafficking to the plasma membrane and stress-induced active release. Altogether, our findings demonstrate a vesicular trafficking system bringing excess intracellular Hsp70 to fulfill its immunostimulatory role. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C222.

Endothelial Progenitor Cells: Quod Erat Demonstrandum?

A constantly growing body of research has been performed mainly over the past decade and a half, aiming to facilitate our still limited understanding of the biology and function of various different cell types which collectively have been termed endothelial progenitor cells. This review intends to critically outline aspects of terminology, origin and applicability of the somatic progenitors which are thought to maintain integrity and support regeneration of the inner lining of vessels through proliferation. Despite reasonable progress in the field, we are still not confident regarding the precise phenotype and function of endothelial progenitor cells. Model systems that have been developed to study the vascular regenerative potential of human endothelial progenitor cells in animal models in vivo present principle tools for testing new agents that modulate vascular homeostasis and regeneration.

Stem Cells Review Series

Cell therapy holds great promise for the treatment of cardiovascular disease. For the past 2 decades, investigators have been developing methods to implant various types of cells to heal the heart and improve the recovery of the heart after ischemia. Although multiple experimental and several clinical studies showed that different subsets of bone marrow–derived cells and other adult progenitor cells improved the recovery of heart function, the mechanism of action and the extent to which adult progenitor cells are able to restore cardiac function in end-stage heart failure is debated extensively. Therefore, this review series will summarize the current status of cell therapy in clinical trials and discuss the potential of different cell types and ways to enhance their functions. Additionally, epigenetic molecular mechanisms that regulate stem cell functions and fate decisions will be summarized. The first evidence that cell therapy may constitute a therapeutic option for the treatment of ischemic disease was provided by Asahara et al in 1997, who showed that bone marrow–derived CD34+ cells can give rise to endothelial-like cells and improve neovascularization after ischemia.1 In attempts to replace cardiomyocytes, initial animal studies used isolated neonatal cardiomyocytes to replace dead muscle tissue. Because of the limited availability of mature human cardiomyocytes, skeletal muscle cells were considered as an alternative cell source, which indeed improved cardiac function after transplantation of the cells in animal models. Subsequent clinical trials showed some benefit but also raised safety concerns with respect to arrhythmic events induced by nonintegrated skeletal muscle cells.2 Cultured cardiac stem cells, …

Pretreatment with Interferon-γ Enhances the Therapeutic Activity of Mesenchymal Stromal Cells in Animal Models of Colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohn's disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon-γ (IFN-γ). In this article, we assessed the effects of IFN-γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN-γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)- and trinitrobenzene sulfonate (TNBS)-induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS-induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS-induced colitis. IMSC-treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN-γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN-γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo.

Intraspinal rewiring of the corticospinal tract via BDNF-TrkB signaling after brain injury

Brain-derived neurotrophic factor (BDNF) regulates neural cell survival mainly by activating TrkB receptors. Several lines of evidence support a key role for BDNF-TrkB signaling in survival of adult retinal ganglion cells in animal models of optic nerve injury (ONI), but the neuroprotective effect of exogenous BDNF is transient. Glial cells have recently attracted considerable attention as mediators of neural cell survival, and TrkB expression in retinal glia suggests its role in neuroprotection. To elucidate this point directly, we examined the effect of ONI on TrkBflox/flox:glial fibrillary acidic protein (GFAP)–Cre+ (TrkBGFAP) knockout (KO) mice, in which TrkB is deleted in retinal glial cells. ONI markedly increased mRNA expression levels of basic fibroblast growth factor (bFGF) in wild-type (WT) mice but not in TrkBGFAP KO mice. Immunohistochemical analysis at 7 days after ONI (d7) revealed bFGF up-regulation mainly occurred in Müller glia. ONI-induced retinal ganglion cell loss in WT mice was consistently mild compared with TrkBGFAP KO mice at d7. On the other hand, ONI severely decreased TrkB expression in both WT and TrkBGFAP KO mice after d7, and the severity of retinal degeneration was comparable with TrkBGFAP KO mice at d14. Our data provide direct evidence that glial TrkB signaling plays an important role in the early stage of neural protection after traumatic injury.

Complement mediated apoptosis leads to the loss of retinal ganglion cells in animal model of glaucoma

This study investigated the role of complement in the protection of retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma. Intraocular pressure (IOP) was elevated in the right eye of Lewis rats by laser photocoagulation (two treatments, 7days apart) of episcleral and limbal veins. Left eye did not receive laser treatment and served as control. Animals were injected with cobra venom factor every fifth day starting day 7 after first laser, to deplete the complement system. Animals were sacrificed at 6-week post-laser. Levels of C3 split products and membrane attack complex (MAC) were elevated in the retina of eyes with increased IOP and complement depletion reduced the loss of Brn3a+ RGCs accompanied by decreased expression of GFAP and reduced MAC deposition. In complement depleted rats with increased IOP, reduced TUNEL+ cells in ganglion cell layer, and decreased levels of active caspase-8 and active caspase-9 was observed compared to PBS treated complement sufficient rats with increased IOP. Interestingly, complement depletion also resulted in reduction of calcium influx and levels of BAD in the retinal cells of the eyes with increased IOP. Together, our results provide evidence that complement mediated apoptosis plays a pivotal role in the loss of RGCs in chronic ocular hypertension model of glaucoma.

Mycophenolate mofetil (MMF) does not slow the progression of subclinical atherosclerosis in SLE over 2 years.

Accelerated atherosclerosis is a major cause of mortality in SLE. Mycophenolate mofetil (MMF) has been shown to suppress growth factor-induced proliferation of vascular smooth muscle and endothelial cells in animal models. We hypothesized that MMF might modify the inflammatory component of atherosclerosis in SLE. We examined the effect of MMF on atherosclerosis as measured by changes in carotid intima-media thickness (IMT) or coronary artery calcium (CAC) over 2 years. CAC and carotid IMT were measured at baseline and 2 years later in a cohort of 187 patients with SLE. The cohort was 91% women, 59% Caucasian, and 35% African-American, with a mean age of 45 ± 11 years. Of these, 12.5% (n = 25) received MMF during follow-up. The daily dose ranged from 500 to 3,000 mg/day, and duration ranged from 84 days to the entire 2 years. We divided MMF users into three groups: low exposure (<1,500 mg average daily dose), high exposure (≥1,500 average daily dose), and any exposure of MMF (<1,500 or ≥1,500 average daily dose) for 2 years. The mean CAC increased in all four groups: no MMF: 1.17-1.28, low MMF: 1.02-1.13, high MMF: 1.44-1.61, and any MMF: 1.21-1.34 log-Agatston units. Compared to no MMF, there was no statistically different change between the three groups (p = 0.99, 0.87, and 0.91). Similarly, mean carotid IMT increased in all four groups: no MMF: 0.58-0.66, low MMF: 0.55-0.60, high MMF: 0.56-0.71, and any MMF: 0.56-0.66. We then adjusted for statin use, lupus nephritis, body mass index, systolic blood pressure, cholesterol, and age during the 2-year follow-up. The association between MMF exposure and change in CAC or carotid IMT was not statistically significant (p = 0.63 for CAC, and p = 0.085 for carotid IMT). There was no evidence that MMF slowed or decreased the progression of atherosclerosis as measured by carotid IMT or CAC. Because the number of patients taking MMF was only twenty-five, larger studies for longer time periods are needed to explore any effect of MMF on subclinical atherosclerosis in SLE.

Systemic administration of naked plasmid encoding HGF attenuates puromycin aminonucleoside-induced damage of murine glomerular podocytes

Podocyte injury is considered to play important roles in the pathogenesis of human glomerular disease. There is accumulating evidence suggesting that hepatocyte growth factor (HGF) elicits preventive activity for glomerular cells in animal models of chronic renal diseases. In this study, we demonstrated that delivery of a naked plasmid vector encoding the human HGF gene into mice by a hydrodynamic-based in vivo gene transfection approach markedly reduced proteinuria and attenuated podocyte injury in a mouse model induced by puromycin aminonucleoside (PAN) injection. Systemic administration by rapid injection via the tail vein of a naked plasmid containing HGF cDNA driven under a cytomegalovirus promoter (pCMV-HGF) produced a remarkable level of human HGF protein in the circulation. Tissue distribution studies suggested that the kidney expressed a high level of the HGF transgene. Meanwhile, compared with tubules and interstitium, a higher level of exogenous HGF protein was detected in the glomeruli. Administration of pCMV-HGF dramatically abated the urine albumin excretion and podocyte injury in PAN nephropathy in mice. Exogenous expression of HGF produced evidently beneficial effects, leading to restoration of Wilms' tumor-1 (WT1) and α-actinin-4 expression and attenuation of ultrastructural damage of the podocytes. In vitro, HGF not only restored WT1 and α-actinin-4 expression but also inhibited albumin leakage of podocytes incubated with PAN in a Transwell culture chamber. These results suggest that HGF might provide a novel strategy for amelioration of podocyte injury.

The monoclonal antibody Zt/f2 targeting RON receptor tyrosine kinase as potential therapeutics against tumor growth-mediated by colon cancer cells

BackgroundOverexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb) Zt/f2 in experimental cancer therapy.ResultsZt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED50 = 2.3 nmol/L). Receptor binding studies revealed that Zt/f2 interacts with an epitope(s) located in a 49 amino acid sequence coded by exon 11 in the RON β-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo.ConclusionsZt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.

Uso de las células troncales embrionarias en el tratamiento de diversas enfermedades en el humano

Las células troncales (CT) también llamadas células madre tienen dos características que las distinguen de las demás células de un organismo: son células no especializadas que pueden auto-renovarse mediante la mitosis y en ciertas condiciones fisiológicas o experimentales pueden diferenciarse a células con una función específica como neuronas, hepatocitos, células del cartílago, etc. (McKay, 2000). Se han descrito dos tipos de CT, tanto en los animales como en el ser humano: las CT embrionarias (CTE) y obtenidas de tejido adulto. Ambas tienen características y funciones diferentes.

Specification of Sensory Fates in the Vertebrate Inner Ear

The otic placode gives rise to the inner sensory organs subserving hearing and balance, along with the associated auditory and vestibular ganglion neurons that supply their afferent innervation. Our lab is using both chicken and zebrafish to study the molecular basis of cell fate specification within the otic neurosensory lineages. In chicken embryos, Wnt/β-catenin signaling can promote vestibular over auditory sensory organ fates at early stages if the pathway is manipulated by retrovirus-mediated overexpression. As the organs begin to differentiate into hair cells and supporting cells, the miR-183 family of microRNAs can control the number of HCs that form in the zebrafish, as shown by gene knockdown and overexpression. We are currently studying whether these microRNAs play a similar role in the chicken ear. Somewhat later in development, Wnt overexpression can influence the differentiation of distinct hair cell types (tall vs. short) in the auditory sensory organ of the bird. These findings may offer therapeutic strategies to induce the regeneration of supporting cells into the appropriate types of hair cells in animal models of deafness.

Seizure‐induced neurogenesis in the adult brain

It is well established that seizures increase adult neurogenesis in the subventricular and subgranular zones, the most neurogenic regions of the adult rodent and apparently human brain. However, the role of increased neurogenesis in these areas in seizure generation (ictogenesis) and epileptogenesis remains elusive. It is of utmost importance to explore how the cells that are born in response to epileptic seizures are functionally integrated into the existing neuronal networks, and how this integration would contribute to the excitability of this network. This will determine whether increased neurogenesis is beneficial or counteractive to ictogenesis and epileptogenesis. Some of the crucial factors affecting the functional integration of newborn cells seem to be excessive neuronal activity and/or inflammatory microenvironment, both associated with acute, as well as chronic, epileptic conditions. This review will focus on aspects of the functional integration of newborn cells in animal models of epilepsy with various degrees of seizure severity and associated microenvironmental alterations in the brain tissue.

Review: Preclinical studies on placenta-derived cells and amniotic membrane: An update

Recent years have seen considerable advances in our knowledge of the biology and properties of stem/progenitor cells isolated from placental tissues. This has encouraged researchers to address the potential effects of these cells in animal models of different diseases, resulting in increasing expectations regarding their possible utility for cell-based therapeutic applications. This rapidly evolving research field is also enriched by studies aimed at expanding the use of the whole amniotic membrane (AM), a well-known surgical material, for pathological conditions other than those tested so far and for which clinical applications already exist. In this review, we provide an update on studies that have been performed with placenta-derived cells and fragments of the entire AM to validate their potential clinical applications in a variety of diseases, in particular those associated with degenerative processes induced by inflammatory and fibrotic mechanisms. We also offer, as far as possible, insight into the interpretation and suggested mechanisms to explain the most important outcomes achieved to date.

Illuminating progression: tracking melanoma metastasis in a gene therapy setting

Illuminating progression: tracking melanoma metastasis in a gene therapy setting

Emodin potentiates the antitumor effects of gemcitabine in pancreatic cancer cells via inhibition of nuclear factor-κB.

Many studies have demonstrated that emodin inhibits the growth and induces the apoptosis and chemo-sensitization of various cancer cells in animal models. The aim of this study was to investigate the molecular mechanism of the chemo-sensitization potential of emodin on gemcitabine in pancreatic cancer cell lines via inhibition of nuclear factor-κB (NF-κB). SW1990 and SW1990/GZ cells were treated with: i) emodin (20 µmol/l), ii) NF-κB inhibitor Bay 11-7082 (5 µmol/l), iii) gemcitabine (20 µmol/l), iv) pre-treated with emodin for 24 h followed by coincubation with gemcitabine for 24 h, or v) pre-treated with Bay 11-7082 for 1 h followed by treatment with gemcitabine for 24 h. SW1990 and SW1990/GZ cells were also treated with emodin (20, 40 and 80 µmol/l). Cellular proliferation and apoptosis were detected by the Cell Counting Kit-8 (CCK-8) assay and flow cytometry. NF-κB protein was detected by Western blotting. SW1990/GZ cell morphological changes were observed under optical and fluorescence microscopes. Emodin strongly inhibited the proliferation and induced the apoptosis of both pancreatic cancer cell lines. Furthermore, emodin combined with gemcitabine induced a higher percentage of growth inhibition and apoptosis in both pancreatic cancer cell lines compared to gemcitabine alone. Pre-treatment of SW1990/GZ cells with Bay 11-7082 for 1 h followed by gemcitabine resulted in greater inhibitory and apoptosis rates compared to gemcitabine alone. The resistant pancreatic cell line SW1990/GZ presented higher constitutive NF-κB protein expression compared to the SW1990 cells. Emodin not only down-regulated NF-κB in a dose-dependent manner in SW1990 and SW1990/GZ cells under unstimulated conditions, but also inhibited gemcitabine-induced NF-κB protein expression. Emodin potentiated the antitumor effects of gemcitabine in pancreatic cancer, which was related to the down-regulation of NF-κB.