Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

Biology and clinical utilization of mesenchymal progenitor cells

Within the complex cellular arrangement found in the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal progenitor cells (MPC). These cells can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into at least seven types of cells: osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes and hematopoietic-supporting stroma. This broad multipotentiality, the feasibility to obtain MPC from bone marrow, cord and peripheral blood and their transplantability support the impact that the use of MPC will have in clinical settings. However, a number of fundamental questions about the cellular and molecular biology of MPC still need to be resolved before these cells can be used for safe and effective cell and gene therapies intended to replace, repair or enhance the physiological function of the mesenchymal and/or hematopoietic systems.

A safety and feasibility study of cell therapy in dilated cardiomyopathy

The aim of this study was to determine if bone marrow mononuclear cell (BMMC) transplantation is safe for moderate to severe idiopathic dilated cardiomyopathy (IDC). Clinical trials have shown that this procedure is safe and effective for ischemic patients, but little information is available regarding non-ischemic patients. Twenty-four patients with IDC, optimized therapy, age 46 ± 11.6 years, 17 males, NYHA classes II-IV, and left ventricular ejection fraction <35% were enrolled in the study. Clinical evaluation at baseline and 6 months after stem cell therapy to assess heart function included echocardiogram, magnetic resonance imaging, cardiopulmonary test, Minnesota Quality of Life Questionnaire, and NYHA classification. After cell transplantation 1 patient showed a transient increase in enzyme levels and 2 patients presented arrhythmias that were reversed within 72 h. Four patients died during follow-up, between 6 and 12 weeks after therapy. Clinical evaluation showed improvement in most patients as reflected by statistically significant decreases in Minnesota Quality of Life Questionnaire (63 ± 17.9 baseline vs 28.8 ± 16.75 at 6 months) and in class III-IV NYHA patients (18/24 baseline vs 2/20 at 6 months). Cardiopulmonary exercise tests demonstrated increased peak oxygen consumption (12.2 ± 2.4 at baseline vs 15.8 ± 7.1 mL·kg-1·min-1 at 6 months) and walked distance (377.2 ± 85.4 vs 444.1 ± 77.9 m at 6 months) in the 6-min walk test, which was not accompanied by increased left ventricular ejection fraction. Our findings indicate that BMMC therapy in IDC patients with severe ventricular dysfunction is feasible and that larger, randomized and placebo-controlled trials are warranted.

Immune potential and differentiation of equine induced pluripotent stem cells (eiPSC)

Induced pluripotent stem cells (iPSC) have the capacity to self renew and differentiate into a myriad of cell types making them potential candidates for cell therapy and regenerative medicine. The goal of this thesis was to determine the characteristics of equine iPSC (eiPSC) that can be harnessed for potential use in veterinary regenerative medicine. Trauma to a horse’s limb often leads to the development of a chronic non-healing wound that lacks a keratinocyte cover, vital to healing. Thus, the overall hypothesis of this thesis was that eiPSC might offer a solution for providing wound coverage for such problematic wounds. Prior to considering eiPSC for clinical applications, their immunogenicity must be studied to ensure that the transplanted cells will be accepted and integrate into host tissues. The first objective of this thesis was to determine the immune response to eiPSC. To investigate the immunogenicity of eiPSC, the expression of major histocompatibility complex (MHC) molecules by the selected lines was determined, then the cells were used in an intradermal transplantation model developed for this study. While transplantation of allogeneic, undifferentiated eiPSC elicited a moderate cellular response in experimental horses, it did not cause acute rejection. This strategy enabled the selection of weakly immunogenic eiPSC lines for subsequent differentiation into lineages of therapeutic importance. Equine iPSC offer a potential solution to deficient epithelial coverage by providing a keratinocyte graft with the ability to differentiate into other accessory structures of the epidermis. The second objective of this thesis was to develop a protocol for the differentiation of eiPSC into a keratinocyte lineage. The protocol was shown to be highly efficient at inducing the anticipated phenotype within 30 days. Indeed, the eiPSC derived vi keratinocytes (eiPSC-KC) showed both morphologic and functional characteristics of primary equine keratinocytes (PEK). Moreover, the proliferative capacity of eiPSC-KC was superior while the migratory capacity, measured as the ability to epithelialize in vitro wounds, was comparable to that of PEK, suggesting exciting potential for grafting onto in vivo wound models. In conclusion, equine iPSC-derived keratinocytes exhibit features that are promising to the development of a stem cell-based skin construct with the potential to fully regenerate lost or damaged skin in horses. However, since eiPSC do not fully escape immune surveillance despite low MHC expression, strategies to improve engraftment of iPSC derivatives must be pursued.

Neural stem cells and cell replacement therapy: making the right cells

The past few years have seen major advances in the field of NSC (neural stem cell) research with increasing emphasis towards its application in cell-replacement therapy for neurological disorders. However, the clinical application of NSCs will remain largely unfeasible until a comprehensive understanding of the cellular and molecular mechanisms of NSC fate specification is achieved. With this understanding will come an increased possibility to exploit the potential of stem cells in order to manufacture transplantable NSCs able to provide a safe and effective therapy for previously untreatable neurological disorders. Since the pathology of each of these disorders is determined by the loss or damage of a specific neural cell population, it may be necessary to generate a range of NSCs able to replace specific neurons or glia rather than generating a generic NSC population. Currently, a diverse range of strategies is being investigated with this goal in mind. In this review, we focus on the relationship between NSC specification and differentiation and discuss how this information may be used to direct NSCs towards a particular fate.

The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (Pcells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

Origin and regenerative potential of vertebrate mechanoreceptor-associated stem cells

Meissner corpuscles and Merkel cell neurite complexes are highly specialized mechanoreceptors present in the hairy and glabrous skin, as well as in different types of mucosa. Several reports suggest that after injury, such as after nerve crush, freeze injury, or dissection of the nerve, they are able to regenerate, particularly including reinnervation and repopulation of the mechanoreceptors by Schwann cells. However, little is known about mammalian cells responsible for these regenerative processes. Here we review cellular origin of this plasticity in the light of newly described adult neural crest-derived stem cell populations. We also discuss further potential multipotent stem cell populations with the ability to regenerate disrupted innervation and to functionally recover the mechanoreceptors. These capabilities are discussed as in context to cellularly reprogrammed Schwann cells and tissue resident adult mesenchymal stem cells.

Interaction of adult human neural crest-derived stem cells with a nanoporous titanium surface is sufficient to induce their osteogenic differentiation

Osteogenic differentiation of various adult stem cell populations such as neural crest-derived stem cells is of great interest in the context of bone regeneration. Ideally, exogenous differentiation should mimic an endogenous differentiation process, which is partly mediated by topological cues. To elucidate the osteoinductive potential of porous substrates with different pore diameters (30 nm, 100 nm), human neural crest-derived stem cells isolated from the inferior nasal turbinate were cultivated on the surface of nanoporous titanium covered membranes without additional chemical or biological osteoinductive cues. As controls, flat titanium without any topological features and osteogenic medium was used. Cultivation of human neural crest-derived stem cells on 30 nm pores resulted in osteogenic differentiation as demonstrated by alkaline phosphatase activity after seven days as well as by calcium deposition after 3 weeks of cultivation. In contrast, cultivation on flat titanium and on membranes equipped with 100 nm pores was not sufficient to induce osteogenic differentiation. Moreover, we demonstrate an increase of osteogenic transcripts including Osterix, Osteocalcin and up-regulation of Integrin β1 and α2 in the 30 nm pore approach only. Thus, transplantation of stem cells pre-cultivated on nanostructured implants might improve the clinical outcome by support of the graft adherence and acceleration of the regeneration process.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

Translational research and therapeutic applications of neural crest-derived stem cells in regenerative periodontology

Regeneration of periodontal tissues aims to utilize tissue engineering techniques to restore lost periodontal tissues including the cementum, periodontal ligament and alveolar bone. Regenerative dentistry and its special field regenerative periodontology represent relatively new and emerging branches of translational stem cell biology and regenerative medicine focusing on replacing and regenerating dental tissues to restore or re-establish their normal function lost during degenerative diseases or acute lesions. The regeneration itself can be achieved through transplantation of autologous or allogenic stem cells, or by improving the tissue self-repair mechanisms (e.g. by application of growth factors). In addition, a combination of stem cells or stem cell-containing tissue with bone implants can be used to improve tissue integration and the clinical outcome. As the oral cavity represents a complex system consisting of teeth, bone, soft tissues and sensory nerves, regenerative periodontology relies on the use of stem cells with relatively high developmental potential. Notably, the potential use of pluripotent stem cell types such as human embryonic stem cells or induced pluripotent stem cells is still aggravated by ethical and practical problems. Thus, other cellular sources such as those readily available in the postnatal craniofacial area and particularly in oral structures offer a much better and realistic alternative as cellular regenerative sources. In this review, we summarize current knowledge on the oral neural crest-derived stem cell populations (oNCSCs) and discuss their potential in regenerative periodontology.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Experimental Basis and New Insights for Cell Therapy in Chronic Obstructive Pulmonary Disease

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multi-potent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.

A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia (Oreochromis niloticus) Model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)