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1.
Methods Mol Biol ; 2206: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754821

RESUMO

Exiting developments in tissue engineering and new insights in stem cell biology have led to new possible strategies for the regeneration of damaged tissues in the oral cavity. The regeneration of the pulp-dentin complex regeneration in particular, has drawn the attention of many researchers because of the high clinical needs. While it is still important to perform in vitro research using a wide variety of cells, scaffolds and growth factors, it is also critical to have a reliable animal model for preclinical trials. In this chapter, we describe a mouse model in which a scaffold resembling a tooth containing dental pulp cells is implanted subcutaneously. We also describe which histological stainings could be used to examine blood vessel formation and the regeneration of the pulp-dentin complex.

2.
J Exp Med ; 217(5)2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32097464

RESUMO

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Macrophages and microglia are crucially involved in the formation and repair of demyelinated lesions. Here we show that myelin uptake temporarily skewed these phagocytes toward a disease-resolving phenotype, while sustained intracellular accumulation of myelin induced a lesion-promoting phenotype. This phenotypic shift was controlled by stearoyl-CoA desaturase-1 (SCD1), an enzyme responsible for the desaturation of saturated fatty acids. Monounsaturated fatty acids generated by SCD1 reduced the surface abundance of the cholesterol efflux transporter ABCA1, which in turn promoted lipid accumulation and induced an inflammatory phagocyte phenotype. Pharmacological inhibition or phagocyte-specific deficiency of Scd1 accelerated remyelination ex vivo and in vivo. These findings identify SCD1 as a novel therapeutic target to promote remyelination.

3.
Transl Stroke Res ; 11(1): 60-79, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31309427

RESUMO

Adult neurogenesis in the subventricular zone is a topic of intense research, since it has vast implications for the fundamental understanding of the neurobiology of the brain and its potential to being harnessed for therapy in various neurological disorders. Investigation of adult neurogenesis has been complicated by the difficulties with characterization of neural stem cells in vivo. However, recent single-cell transcriptomic studies provide more detailed information on marker expression in neural stem cells and their neuronal lineage, which hopefully will result in a more unified discussion. Regulation of the multiple biological steps in adult neurogenesis comprises intrinsic mechanisms as well as extrinsic factors which together orchestrate the process. In this review, we describe the regulating factors and their cellular sources in the physiological condition and provide an overview of the regulating factors mediating stroke-induced stimulation of neurogenesis in the subventricular zone. While there is ongoing debate about the longevity of active post-natal neurogenesis in humans, the subventricular zone has the capacity to upregulate neurogenesis in response to ischemic stroke. Though, the stroke-induced neurogenesis in humans does not seem to translate into adequate functional recovery, which opens discussion about potential treatment strategies to harness this neuroregenerative response. Various therapeutic approaches are explored in preclinical and clinical studies to target endogenous neurogenesis of which some are discussed in this review.

4.
Stem Cells Int ; 2019: 8589149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089335

RESUMO

Pathologies of the central nervous system are characterized by loss of brain tissue and neuronal function which cannot be adequately restored by endogenous repair processes. This stresses the need for novel treatment options such as cell-based therapies that are able to restore damaged tissue or stimulate repair. This study investigated the neuroregenerative potential of the conditioned medium of human dental pulp stem cells (CM-hDPSCs) on neural stem cell (NSC) proliferation and migration as well as on neurite outgrowth of primary cortical neurons (pCNs). Additionally, the effect of leukocyte- and platelet-rich fibrin (L-PRF) priming on the neuroregenerative potential of the hDPSC secretome on NSCs and pCNs was evaluated. L-PRF contains factors that enhance stem cell-induced regeneration, but its effect on hDPSC-mediated neuroregeneration is unknown. This study demonstrated that CM-hDPSCs enhanced neuritogenesis. Moreover, CM-hDPSCs had a chemoattractant effect on NSCs. Although priming hDPSCs with L-PRF increased brain-derived neurotrophic factor secretion, no additional effects on the paracrine-mediated repair mechanisms were observed. These data support the neuroregenerative potential of hDPSCs, and although priming had no additional effect, the potential of L-PRF-primed hDPSCs on distinct regenerative mechanisms remains to be clarified.

5.
Theranostics ; 8(10): 2799-2813, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29774076

RESUMO

Rationale: Pluripotent stem cells (PSCs) are being investigated as a cell source for regenerative medicine since they provide an infinitive pool of cells that are able to differentiate towards every cell type of the body. One possible therapeutic application involves the use of these cells to treat myocardial infarction (MI), a condition where billions of cardiomyocytes (CMs) are lost. Although several protocols have been developed to differentiate PSCs towards CMs, none of these provide a completely pure population, thereby still posing a risk for neoplastic teratoma formation. Therefore, we developed a strategy to (i) monitor cell behavior noninvasively via site-specific integration of firefly luciferase (Fluc) and the human positron emission tomography (PET) imaging reporter genes, sodium iodide symporter (hNIS) and somatostatin receptor type 2 (hSSTr2), and (ii) perform hSSTr2-mediated suicide gene therapy via the clinically used radiopharmacon 177Lu-DOTATATE. Methods: Human embryonic stem cells (ESCs) were gene-edited via zinc finger nucleases to express Fluc and either hNIS or hSSTr2 in the safe harbor locus, adeno-associated virus integration site 1. Firstly, these cells were exposed to 4.8 MBq 177Lu-DOTATATE in vitro and cell survival was monitored via bioluminescence imaging (BLI). Afterwards, hNIS+ and hSSTr2+ ESCs were transplanted subcutaneously and teratomas were allowed to form. At day 59, baseline 124I and 68Ga-DOTATATE PET and BLI scans were performed. The day after, animals received either saline or 55 MBq 177Lu-DOTATATE. Weekly BLI scans were performed, accompanied by 124I and 68Ga-DOTATATE PET scans at days 87 and 88, respectively. Finally, hSSTr2+ ESCs were differentiated towards CMs and transplanted intramyocardially in the border zone of an infarct that was induced by left anterior descending coronary artery ligation. After transplantation, the animals were monitored via BLI and PET, while global cardiac function was evaluated using cardiac magnetic resonance imaging. Results: Teratoma growth of both hNIS+ and hSSTr2+ ESCs could be followed noninvasively over time by both PET and BLI. After 177Lu-DOTATATE administration, successful cell killing of the hSSTr2+ ESCs was achieved both in vitro and in vivo, indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2+ ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed. Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment.


Assuntos
Células-Tronco Embrionárias Humanas/transplante , Infarto do Miocárdio/terapia , Receptores de Somatostatina/metabolismo , Transplante de Células-Tronco/métodos , Teratoma/diagnóstico por imagem , Animais , Linhagem Celular , Feminino , Genes Reporter , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Nus , Infarto do Miocárdio/diagnóstico por imagem , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Octreotida/análogos & derivados , Compostos Organometálicos , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Receptores de Somatostatina/genética , Transplante de Células-Tronco/efeitos adversos , Teratoma/etiologia
7.
Stem Cell Reports ; 10(2): 655-672, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29337119

RESUMO

Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.


Assuntos
Diferenciação Celular/genética , Oligodendroglia/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXE/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/terapia , Antígenos de Superfície/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Proteína Básica da Mielina/genética , Neurônios/patologia , Neurônios/transplante , Oligodendroglia/citologia , Oligodendroglia/transplante , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Transcriptoma/genética
8.
Stem Cells Int ; 2017: 2582080, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29018483

RESUMO

Adequate vascularization, a restricting factor for the survival of engineered tissues, is often promoted by the addition of stem cells or the appropriate angiogenic growth factors. In this study, human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) were applied in an in vivo model of dental pulp regeneration in order to compare their regenerative potential and confirm their previously demonstrated paracrine angiogenic properties. 3D-printed hydroxyapatite scaffolds containing DPSCs and/or SCAPs were subcutaneously transplanted into immunocompromised mice. After twelve weeks, histological and ultrastructural analysis demonstrated the regeneration of vascularized pulp-like tissue as well as mineralized tissue formation in all stem cell constructs. Despite the secretion of vascular endothelial growth factor in vitro, the stem cell constructs did not display a higher vascularization rate in comparison to control conditions. Similar results were found after eight weeks, which suggests both osteogenic/odontogenic differentiation of the transplanted stem cells and the promotion of angiogenesis in this particular setting. In conclusion, this is the first study to demonstrate the successful formation of vascularized pulp-like tissue in 3D-printed scaffolds containing dental stem cells, emphasizing the promising role of this approach in dental tissue engineering.

9.
J Endod ; 43(9S): S12-S16, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28781091

RESUMO

Dental pulp is a highly vascularized and innervated tissue containing a heterogeneous stem cell population with multilineage differentiation potential. Current endodontic treatments focus on the preservation of the pulp tissue and the regeneration of dental pulp after pathological insults. Human dental pulp stem cells (hDPSCs) are currently investigated as stem cell-based therapy for pulp regeneration and for peripheral nerve injury in which neurons and Schwann cells display limited regenerative capacity. We have developed a neuronal differentiation protocol for hDPSCs that requires neurosphere formation before neuronal maturation. Moreover, Schwann cell differentiation of hDPSCs in our group revealed that differentiated hDPSCs have acquired the ability to myelinate and guide neurites from dorsal root ganglia. Besides their dynamic differentiation capacity, hDPSCs were shown to exert a paracrine effect on neural and endothelial cells. Analysis of hDPSC conditioned medium revealed the secretion of a broad spectrum of growth factors including brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, and glial-derived neurotrophic factor. Application of the conditioned medium to endothelial cells promoted cell migration and tubulogenesis, indicating a paracrine proangiogenic effect. This hypothesis was enforced by the enhanced formation of blood vessels in the chorioallantoic membrane assay in the presence of hDPSCs. In addition, transplantation of 3-dimensional-printed hydroxyapatite scaffolds containing peptide hydrogels and hDPSCs into immunocompromised mice revealed blood vessel ingrowth, pulplike tissue formation, and osteodentin deposition suggesting osteogenic/odontogenic differentiation of hDPSCs. Future studies in our research group will focus on the pulp regeneration capacity of hDPSCs and the role of fibroblasts within the pulp extracellular matrix.


Assuntos
Polpa Dentária/citologia , Polpa Dentária/fisiologia , Neovascularização Fisiológica , Neurogênese , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Diferenciação Celular , Humanos , Regeneração
10.
J Nucl Med ; 58(10): 1659-1665, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28596158

RESUMO

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.


Assuntos
Células-Tronco Embrionárias/metabolismo , Endorribonucleases/metabolismo , Edição de Genes , Genes Reporter/genética , Genoma Humano/genética , Tomografia por Emissão de Pósitrons , Dedos de Zinco , Animais , Diferenciação Celular , Linhagem Celular , Endorribonucleases/química , Feminino , Expressão Gênica , Humanos , Fígado/citologia , Camundongos
11.
Cytotherapy ; 19(6): 744-755, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28499585

RESUMO

BACKGROUND AIMS: Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders affecting the normal hematopoietic differentiation process and leading to abnormal maturation and differentiation of all blood cell lineages. Treatment options are limited, and there is an unmet medical need for effective therapies for patients with severe cytopenias. METHODS: We demonstrate that multipotent adult progenitor cells (MAPC) improve the function of hematopoietic progenitors derived from human MDS bone marrow (BM) by significantly increasing the frequency of primitive progenitors as well as the number of myeloid colonies. RESULTS: This effect was more pronounced in a non-contact culture, indicating the importance of soluble factors produced by the MAPC cells. Moreover, the cells did not stimulate the growth of the abnormal MDS clone, as shown by fluorescent in situ hybridization analysis on BM cells from patients with a known genetic abnormality. We also demonstrate that MAPC cells can provide stromal support for patient-derived hematopoietic cells. When MAPC cells were intravenously injected into a mouse model of MDS, they migrated to the site of injury and increased the hematopoietic function in diseased mice. DISCUSSION: The preclinical studies undertaken here indicate an initial proof of concept for the use of MAPC cell therapy in patients with MDS-related severe and symptomatic cytopenias and should pave the way for further investigation in clinical trials.


Assuntos
Células-Tronco Multipotentes/transplante , Síndromes Mielodisplásicas/terapia , Adulto , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Feminino , Hematopoese , Humanos , Hibridização in Situ Fluorescente , Camundongos Endogâmicos C57BL
12.
PLoS One ; 11(12): e0167807, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27936076

RESUMO

Periodontal ligament stem cells (PDLSCs) represent a good source of multipotent cells for cell-based therapies in regenerative medicine. The success rate of these treatments is severely dependent on the establishment of adequate vasculature in order to provide oxygen and nutrients to the transplanted cells. Pharmacological preconditioning of stem cells has been proposed as a promising method to augment their therapeutic efficacy. In this study, the aim was to improve the intrinsic angiogenic properties of PDLSCs by in vitro pretreatment with deferoxamine (DFX; 100µM), fibroblast growth factor-2 (FGF-2; 10ng/mL) or both substances combined. An antibody array revealed the differential expression of several proteins, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). ELISA data confirmed a 1.5 to 1.8-fold increase in VEGF for all tested conditions. Moreover, 48 hours after the removal of DFX, VEGF levels remained elevated (1.8-fold) compared to control conditions. FGF-2 and combination treatment resulted in a 5.4 to 13.1-fold increase in PlGF secretion, whereas DFX treatment had no effect. Furthermore, both PDLSCs as pretreated PDLSCs induced endothelial migration. Despite the significant elevated VEGF levels of pretreated PDLSCs, the induced endothelial migration was not higher by pretreated PDLSCs. We find that the observed induced endothelial cell motility was not dependent on VEGF, since blocking the VEGFR1-3 with Axitinib (0.5nM) did not inhibit endothelial motility towards PDLSCs. Taken together, this study provides evidence that preconditioning with DFX and/or FGF-2 significantly improves the angiogenic secretome of PDLSCs, in particular VEGF and PlGF secretion. However, our data suggest that VEGF is not the only player when it comes to influencing endothelial behavior by the PDLSCs.


Assuntos
Desferroxamina/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Adolescente , Adulto , Indutores da Angiogênese/farmacologia , Animais , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Adulto Jovem
13.
Adv Exp Med Biol ; 951: 199-235, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27837566

RESUMO

Over the past decade, dental tissues have become an attractive source of mesenchymal stem cells (MSCs). Dental stem cells (DSCs) are not only able to differentiate into adipogenic, chondrogenic and osteogenic lineanges, but an increasing amount of research also pointed out their potential applicability in numerous clinical disorders, such as myocardial infarction, neurodegenerative diseases and diabetes. Together with their multilineage differentiation capacity, their easy availability from extracted third molars makes these stem cells a suitable alternative for bone marrow-derived MSCs. More importantly, DSCs appear to retain their stem cell properties following cryopreservation, a key aspect in their long-term preservation and upscale production. However, the vast number of different cryopreservation protocols makes it difficult to draw definite conclusions regarding the behavior of these stem cells. The routine application and banking of DSCs is also associated with some other pitfalls, such as interdonor variability, cell culture-induced changes and the use of animal-derived culture medium additives. Only thorough assessment of these challenges and the implementation of standardized, GMP procedures will successfully lead to better treatment options for patients who no longer benefit from current stem cell therapies.


Assuntos
Bancos de Espécimes Biológicos/organização & administração , Criopreservação/métodos , Polpa Dentária/citologia , Células Secretoras de Insulina/citologia , Miócitos Cardíacos/citologia , Neurônios/citologia , Células-Tronco/citologia , Diferenciação Celular , Proliferação de Células , Crioprotetores/farmacologia , Meios de Cultura/farmacologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/fisiologia , Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Dimetil Sulfóxido/farmacologia , Humanos , Células Secretoras de Insulina/fisiologia , Células Secretoras de Insulina/transplante , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Neurônios/fisiologia , Neurônios/transplante , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
14.
Stem Cells Int ; 2016: 9762871, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27688777

RESUMO

Within the field of tissue engineering, natural tissues are reconstructed by combining growth factors, stem cells, and different biomaterials to serve as a scaffold for novel tissue growth. As adequate vascularization and innervation are essential components for the viability of regenerated tissues, there is a high need for easily accessible stem cells that are capable of supporting these functions. Within the human tooth and its surrounding tissues, different stem cell populations can be distinguished, such as dental pulp stem cells, stem cells from human deciduous teeth, stem cells from the apical papilla, dental follicle stem cells, and periodontal ligament stem cells. Given their straightforward and relatively easy isolation from extracted third molars, dental stem cells (DSCs) have become an attractive source of mesenchymal-like stem cells. Over the past decade, there have been numerous studies supporting the angiogenic, neuroprotective, and neurotrophic effects of the DSC secretome. Together with their ability to differentiate into endothelial cells and neural cell types, this makes DSCs suitable candidates for dental tissue engineering and nerve injury repair.

15.
Med Res Rev ; 36(6): 1080-1126, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27439773

RESUMO

Stroke is the second most common cause of death and is a major cause of permanent disability. Given the current demographic trend of an ageing population and associated increased risk, the prevalence of and socioeconomic burden caused by stroke will continue to rise. Current therapies are unable to sufficiently ameliorate the disease outcome and are not applicable to all patients. Therefore, strategies such as cell-based therapies with mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPSC) pave the way for new treatment options for stroke. These cells showed great preclinical promise despite the fact that the precise mechanism of action and the optimal administration route are unknown. To gain dynamic insights into the underlying repair processes after stem cell engraftment, noninvasive imaging modalities were developed to provide detailed spatial and functional information on the donor cell fate and host microenvironment. This review will focus on MSCs and iPSCs as types of widely used stem cell sources in current (bio)medical research and compare their efficacy and potential to ameliorate the disease outcome in animal stroke models. In addition, novel noninvasive imaging strategies allowing temporospatial in vivo tracking of transplanted cells and coinciding evaluation of neuronal repair following stroke will be discussed.


Assuntos
Isquemia Encefálica/terapia , Transplante de Células-Tronco/métodos , Acidente Vascular Cerebral/terapia , Animais , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Medições Luminescentes/métodos , Imagem por Ressonância Magnética/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Tomografia por Emissão de Pósitrons/métodos , Regeneração/fisiologia , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia , Tomografia Computadorizada de Emissão de Fóton Único/métodos
16.
Stem Cell Reports ; 5(6): 1183-1195, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26626179

RESUMO

Muscular dystrophies are a heterogeneous group of myopathies, characterized by muscle weakness and degeneration, without curative treatment. Mesoangioblasts (MABs) have been proposed as a potential regenerative therapy. To improve our understanding of the in vivo behavior of MABs and the effect of different immunosuppressive therapies, like cyclosporine A or co-stimulation-adhesion blockade therapy, on cell survival noninvasive cell monitoring is required. Therefore, cells were transduced with a lentiviral vector encoding firefly luciferase (Fluc) and the human sodium iodide transporter (hNIS) to allow cell monitoring via bioluminescence imaging (BLI) and small-animal positron emission tomography (PET). Non-H2 matched mMABs were injected in the femoral artery of dystrophic mice and were clearly visible via small-animal PET and BLI. Based on noninvasive imaging data, we were able to show that co-stim was clearly superior to CsA in reducing cell rejection and this was mediated via a reduction in cytotoxic T cells and upregulation of regulatory T cells.


Assuntos
Vasos Sanguíneos/citologia , Desenvolvimento Muscular , Distrofia Muscular Animal/terapia , Tomografia por Emissão de Pósitrons/métodos , Transplante de Células-Tronco , Células-Tronco/citologia , Simportadores/análise , Animais , Linhagem Celular , Sobrevivência Celular , Ciclosporina/uso terapêutico , Genes Reporter , Humanos , Imunossupressores/uso terapêutico , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Medições Luminescentes , Camundongos Endogâmicos C57BL , Camundongos Nus , Distrofia Muscular Animal/diagnóstico , Distrofia Muscular Animal/patologia , Imagem Óptica , Simportadores/genética , Transdução Genética
17.
Stem Cell Reports ; 5(5): 918-931, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26455413

RESUMO

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.


Assuntos
Células-Tronco Embrionárias/metabolismo , Marcação de Genes/métodos , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Recombinases/metabolismo , Transgenes , Células Cultivadas , Metilação de DNA , Dependovirus/genética , Células-Tronco Embrionárias/citologia , Inativação Gênica , Loci Gênicos , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Recombinases/genética
18.
Stem Cell Rev Rep ; 11(2): 254-74, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25534590

RESUMO

The interest in the use of stem cells as a source for therapy has increased dramatically over the last decades. Different stem cell types have been tested in both in vitro and in vivo models, because of their properties such as differentiation potential, trophic effects and immune modulatory properties. To further optimize the use of different stem cell types for the treatment of disease in a clinical setting, it is necessary to know more about the in vivo behavior of these cells following engraftment. Until now, the golden standard to preclinically evaluate cell therapy was histology, which is an invasive method as the animals need to be sacrificed. This hampers the generation of dynamic information and results in only one single point in time available for analysis per animal. For more information regarding cell migration, in situ persistence, viability, proliferation and differentiation, molecular imaging can be used for imaging cells after transplantation dynamically and longitudinally, in a noninvasive way. With this technology, it becomes possible to track cells within the same subjects over a long period of time.


Assuntos
Diferenciação Celular/genética , Rastreamento de Células/métodos , Imagem Molecular/métodos , Células-Tronco/citologia , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Radioisótopos , Transplante de Células-Tronco
19.
PLoS One ; 9(4): e94833, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24747914

RESUMO

PURPOSE: The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters. METHODS: First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, 124I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using 99mTcO4- radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell's differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI. RESULTS: The expression of both imaging reporter genes was functional and specific. An elution of 99mTcO4- from the cells was observed, with 31% retention after 3 h. After labeling cells with 124I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using 124I small-animal PET, CLI and BLI. CONCLUSIONS: This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.


Assuntos
Luminescência , Células-Tronco Mesenquimais/metabolismo , Imagem Multimodal/métodos , Imagem Óptica/métodos , Tomografia por Emissão de Pósitrons/métodos , Simportadores/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Genes Reporter/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Radioisótopos do Iodo , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Fator 1 de Elongação de Peptídeos/genética , Puromicina/farmacologia
20.
J Nucl Med ; 54(3): 447-54, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23353687

RESUMO

UNLABELLED: Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). METHODS: Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET. RESULTS: The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells. CONCLUSION: MSCs and MAPCs can be successfully labeled with (18)F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells' ultrastructural properties are not influenced by (18)F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with (18)F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.


Assuntos
Células-Tronco Adultas/diagnóstico por imagem , Fluordesoxiglucose F18 , Células-Tronco Mesenquimais/diagnóstico por imagem , Células-Tronco Multipotentes/diagnóstico por imagem , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Diferenciação Celular , Células Cultivadas , Radioisótopos de Flúor , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/ultraestrutura , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Ratos , Medicina Regenerativa , Engenharia Tecidual
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