RESUMEN
The esophagus is a tubular organ with a multi-laminated tissue structure that functions to transport nutrition from the oral cavity to the stomach. Several diseases of the esophagus including congenital disorders require complete surgical esophagectomy. Ideally, segmental removal of the diseased/damaged tissue would spare the unaffected tissue and preserve organ function. To this end, a novel tissue engineered implant, the CellspanTM Esophageal Implant (CEI) was used to repair the esophagus following segmental resection of the thoracic esophagus in a porcine model. The current study investigated the mechanical strength and the associated tissue architecture of the CEI-stimulated tissue. The CEI bridged the proximal and distal native esophageal ends to restore the conduit by stimulating a regeneration process that progressed from a fibrovascular scar at 30-days to a fully epithelialized lumen at 90-days, followed by submucosal regeneration and regeneration of a 'laminated' adventitia with smooth muscle development in the 365-day cohort. The mechanical strength of the newly developed tissue as well as the flanking native tissue were assessed using a probe-burst pressure test (ASTM D6797-15). The burst pressures at all three time points were comparable to the native tissue flanking the implant. In addition, the overall pressure required to burst through both the native and regenerated tissues increased with increasing time post-implantation.
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Esófago , Ingeniería de Tejidos , Animales , Fenómenos Biomecánicos , Esofagectomía , Esófago/fisiología , Humanos , Regeneración , PorcinosRESUMEN
Diseases of the esophagus, damage of the esophagus due to injury or congenital defects during fetal esophageal development, i.e., esophageal atresia (EA), typically require surgical intervention to restore esophageal continuity. The development of tissue engineered tubular structures would improve the treatment options for these conditions by providing an alternative that is organ sparing and can be manufactured to fit the exact dimensions of the defect. An autologous tissue engineered Cellspan Esophageal ImplantTM (CEI) was surgically implanted into piglets that underwent surgical resection of the esophagus. Multiple survival time points, post-implantation, were analyzed histologically to understand the tissue architecture and time course of the regeneration process. In addition, we investigated CT imaging as an "in-life" monitoring protocol to assess tissue regeneration. We also utilized a clinically relevant animal management paradigm that was essential for long term survival. Following implantation, CT imaging revealed early tissue deposition and the formation of a contiguous tissue conduit. Endoscopic evaluation at multiple time points revealed complete epithelialization of the lumenal surface by day 90. Histologic evaluation at several necropsy time points, post-implantation, determined the time course of tissue regeneration and demonstrated that the tissue continues to remodel over the course of a 1-year survival time period, resulting in the development of esophageal structural features, including the mucosal epithelium, muscularis mucosae, lamina propria, as well as smooth muscle proliferation/migration initiating the formation of a laminated adventitia. Long term survival (1 year) demonstrated restoration of oral nutrition, normal animal growth and the overall safety of this treatment regimen.
RESUMEN
INTRODUCTION: Resection and reconstruction of the esophagus remains fraught with morbidity and mortality. Recently, data from a porcine reconstruction model revealed that segmental esophageal reconstruction using an autologous mesenchymal stromal cell-seeded polyurethane graft (Cellspan esophageal implant [CEI]) can facilitate esophageal regrowth and regeneration. To this end, a patient requiring a full circumferential esophageal segmental reconstruction after a complex multiorgan tumor resection was approved for an investigational treatment under the Food and Drug Administration Expanded Access Use (Investigational New Drug 17402). METHODS: Autologous adipose-derived mesenchymal stromal cells (Ad-MSCs) were isolated from the Emergency Investigational New Drug patient approximately 4 weeks before surgery from an adipose tissue biopsy specimen. The Ad-MSCs were grown and expanded under current Good Manufacturing Practice manufacturing conditions. The cells were then seeded onto a polyurethane fiber mesh scaffold (Cellspan scaffold) and cultured in a custom bioreactor to manufacture the final CEI graft. The cell-seeded scaffold was then shipped to the surgical site for surgical implantation. After removal of a tumor mass and a full circumferential 4 cm segment of the esophagus that was invaded by the tumor, the CEI was implanted by suturing the tubular CEI graft to both ends of the remaining native esophagus using end-to-end anastomosis. RESULTS: In this case report, we found that a clinical-grade, tissue-engineered esophageal graft can be used for segmental esophageal reconstruction in a human patient. This report reveals that the graft supports regeneration of the esophageal conduit. Histologic analysis of the tissue postmortem, 7.5 months after the implantation procedure, revealed complete luminal epithelialization and partial esophageal tissue regeneration. CONCLUSIONS: Autologous Ad-MSC seeded onto a tubular CEI tissue-engineered graft stimulates tissue regeneration following implantation after a full circumferential esophageal resection.
RESUMEN
The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3-dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ-induced IDO production in MSC. Chemical phosphoinositide 3-kinase (PI3K) pan-inhibition, PI3Kα-specific inhibition or shRNA knockdown diminished IFNγ-induced IDO production. This effect involved PI3Kα-mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2-like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC-mediated T-cell suppression. The same effect could be induced using short-term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype.
Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Transcripción STAT1/metabolismo , Fosfatidilinositol 3-Quinasa Clase I , Regulación hacia Abajo , Humanos , Interferón gamma/metabolismo , Factor de Transcripción STAT3/metabolismo , Regulación hacia ArribaRESUMEN
Clinical trials investigating mesenchymal stromal cell (MSC) therapy for bronchopulmonary dysplasia have been initiated; however, the optimal delivery route and functional effects of MSC therapy in newborns remain incompletely established. We studied the morphologic and functional effects of intranasal versus i.p. MSC administration in a rodent model of neonatal lung injury. Cultured human cord tissue MSCs (0.1, 0.5, or 1 × 10(6) cell per pup) were given intranasally or i.p. to newborn severe combined immunodeficiency-beige mice exposed to 90% O2 from birth; sham controls received an equal volume of phosphate-buffered saline. Lung mechanics, engraftment, lung growth, and alveolarization were evaluated 8 weeks after transplantation. High-dose i.p. MSC administration to newborn mice exposed to 90% O2 resulted in the restoration of normal lung compliance, elastance, and pressure-volume loops (tissue recoil). Histologically, high-dose i.p. MSC administration was associated with alveolar septal widening, suggestive of interstitial matrix modification. Intranasal MSC or lower-dose i.p. administration had no significant effects on lung function or alveolar remodeling. Pulmonary engraftment was rare in all the groups. These findings suggest that high-dose systemic administration of human cultured MSCs can restore normal compliance in neonatally injured lungs, possibly by paracrine modulation of the interstitial matrix. Intranasal delivery had no obvious pulmonary effects.
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Lesión Pulmonar/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Cordón Umbilical/metabolismo , Administración Intranasal , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/metabolismo , Linaje de la Célula , Células Cultivadas , Humanos , Hiperoxia , Inyecciones Intraperitoneales , Pulmón/patología , Ratones , Ratones SCID , Oxígeno/químicaRESUMEN
BACKGROUND: Stem cell therapy may help restore cardiac function after acute myocardial infarction (AMI), but the optimal therapeutic cell type has not been identified. METHODS: We examined the effects of CD34-/CD45- human unrestricted somatic stem cells (USSCs) in pigs (n = 30) with an AMI created by a 90-min occlusion of the left anterior descending coronary artery. Pigs were randomly assigned to receive either USSCs (302 ± 23 × 10(6) cells) or phosphate-buffered saline via 15 NOGA-guided transendocardial injections 10 days after AMI. Cyclosporine A (10 mg/kg orally, twice a day) was started in all pigs 3 days before control or cell treatment. Cardiac function was assessed by echocardiography before injection and at 4 and 8 weeks after treatment. Serum titers for pig IgG antibodies against USSCs were also measured at these time points and before AMI. RESULTS: Compared with control pigs, USSC-treated pigs showed no significant differences in any of the functional parameters examined. USSC-treated pigs showed variable increases in anti-USSC IgG antibody titers in the blood and chronic inflammatory infiltrates at the cell injection sites. Immunohistochemical studies of the injection sites using human anti-mitochondrial antibodies failed to detect implanted USSCs. CONCLUSIONS: We conclude that human USSCs did not improve cardiac function in a pig model of AMI. Cell transplantation in a xenogeneic setting may obscure the benefits of stem cell therapy.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Infarto del Miocardio/terapia , Células Madre Pluripotentes/trasplante , Trasplante Heterólogo/métodos , Animales , Anticuerpos Heterófilos/sangre , Anticuerpos Heterófilos/inmunología , Antígenos Heterófilos/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Pruebas de Función Cardíaca , Humanos , Inmunosupresores/uso terapéutico , Mitocondrias/inmunología , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Células Madre Pluripotentes/citología , Distribución Aleatoria , Sus scrofa , Trasplante Heterólogo/inmunología , Insuficiencia del TratamientoRESUMEN
BACKGROUND: TNF and its receptors TNF-Receptor 1 (TNFR1, CD120a) and TNF-Receptor 2 (TNFR2, CD120b) have been implicated in the rejection of transplanted cells and organs. Although pig TNFR1 (pTNFR1) is known to mediate the effects of human TNF in a xenogeneic setting, it is unclear whether pig TNFR2 (pTNFR2) could contribute to xenograft rejection. METHODS: We have cloned the cDNA of various pTNFR2 variants by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. We have characterized the predicted proteins with bioinformatic tools and conducted expression, affinity, and functional studies to investigate their roles. RESULTS: We have identified four isoforms of pTNFR2: one comprising the four cysteine-rich domains (CRD) conserved between species, a shorter variant (pTNFR2ΔE7-10) encoding for a soluble isoform, another with only three CRD due to the lack of exon 4 (pTNFR2ΔE4), and a fourth variant containing both modifications. Accordingly, multiple mRNA transcripts were observed by northern blotting. Quantitative RT-PCR determined high pTNFR2 expression in lung and immune cells and detected the two alternative splicings in all cells/tissues examined. The full receptor was moderately expressed on the surface of pig cells such as porcine aortic endothelial cells and PK-15 and was regulated by TNF. On the contrary, the membrane-bound pTNFR2ΔE4 was located only intracellularly. Plasmon resonance studies showed that pTNFR2 binds pig and human TNFα with high affinity, but pTNFR2ΔE4 interacts poorly with pig TNFα and does not bind to the human cytokine. Moreover, pull-down experiments with the two recombinant soluble isoforms consistently demonstrated that the two bound together and soluble pTNFR2ΔE4 was able to modulate the TNF inhibitory activity of pTNFR2-GST in a cell-based assay. CONCLUSION: The pTNFR2 may participate in the process of xenograft rejection and other related events, as well as be used in soluble form to block TNF in this setting. In addition, we have discovered other pTNFR2 isoforms that may affect the pig immune responses and have an impact on rejection of xenografts.
Asunto(s)
Membrana Celular/metabolismo , Rechazo de Injerto/fisiopatología , Receptores Tipo II del Factor de Necrosis Tumoral/análisis , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Trasplante Heterólogo/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Riñón/citología , Riñón/metabolismo , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas , ARN Mensajero/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , PorcinosRESUMEN
BACKGROUND: Extensive studies in rodents have identified olfactory ensheathing cells (OECs) as promising candidates for cell-based therapies of spinal cord and peripheral nerve injury. Previously, we demonstrated that short-term cultured adult porcine OECs can remyelinate the rodent and non-human primate spinal cord. Here, we studied the impact of the culturing interval on the remyelinating capacity of adult porcine OECs. METHODS: Cells were maintained for 1, 2, and 4 to 6 weeks in vitro prior to transplantation into the demyelinated rat spinal cord. Parallel to this, the in vitro phenotypic properties of the OEC preparations used for transplantation were analyzed with regard to morphology, low affinity nerve growth factor receptor (p75(NTR)) expression and proliferation. RESULTS: We report that prolonged culturing of adult porcine OECs resulted in impaired remyelination of the adult rat spinal cord. Animals receiving transplants of OECs maintained in vitro for 2 weeks displayed significantly less remyelinated axons than those animals that received OEC transplants cultured for 1 week. There was virtually no remyelination after transplantation of OECs cultured for 4 to 6 weeks. The adult porcine OECs displayed a progressive lost of p75(NTR)-expression as determined by immunostaining and flow cytometry with time in culture. CONCLUSIONS: Taken together, the results indicate that porcine OECs undergo systematic changes with time in culture that result in reduced p75(NTR)-expression, decreased proliferation, and reduced remyelinating capability with time in vitro indicating that relatively short term cultures with limited expansion would be required for transplantation studies.
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Trasplante de Células/métodos , Vaina de Mielina/metabolismo , Regeneración Nerviosa/fisiología , Vías Olfatorias/citología , Médula Espinal/fisiología , Animales , Técnicas de Cultivo de Célula , Forma de la Célula , Células Cultivadas , Humanos , Fenotipo , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/metabolismo , Médula Espinal/patología , Médula Espinal/ultraestructura , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Porcinos , Factores de TiempoRESUMEN
OBJECTIVE: Unrestricted somatic stem cells (USSCs) were successfully identified from human cord blood. However, the efficacy of USSC transplantation for improving left ventricular (LV) function post myocardial infarction (MI) is still controversial. METHODS AND RESULTS: PBS, 1x10(6) human fibroblasts (Fbr), 1x10(5) USSCs (LD), or 1x10(6) USSCs (HD) were transplanted intramyocardially 20 minutes after ligating the LAD of nude rats. Echocardiography and a microtip conductance catheter at day 28 revealed a dose-dependent improvement of LV function after USSC transplantation. Necropsy examination revealed dose-dependent augmentation of capillary density and inhibition of LV fibrosis. Dual-label immunohistochemistry for cardiac troponin-I and human nuclear antigen (HNA) demonstrated that human cardiomyocytes (CMCs) were dose-dependently generated in ischemic myocardium 28 days after USSC transplantation. Similarly, dual-label immunostaining for smooth muscle actin and class I human leukocyte antigen or that for von Willebrand factor and HNA also revealed a dose-dependent vasculogenesis after USSC transplantation. RT-PCR indicated that expression of human-specific genes of CMCs, smooth muscle cells, and endothelial cell markers in infarcted myocardium were significantly augmented in USSC-treated animals compared with control groups. CONCLUSIONS: USSC transplantation leads to functional improvement and recovery from MI and exhibits a significant and dose-dependent potential for concurrent cardiomyogenesis and vasculogenesis.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Circulación Coronaria/fisiología , Infarto del Miocardio/terapia , Células Madre Pluripotentes/trasplante , Remodelación Ventricular/fisiología , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Sangre Fetal/citología , Humanos , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Distribución Aleatoria , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Función Ventricular/fisiologíaRESUMEN
Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT-derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling. We hypothesized that SNF2-type ATP-dependent chromatin factors contribute to epigenetic reprogramming and the relative amount of these factors in the donor cell affects developmental potential of the reconstructed embryos. In order to test this hypothesis, we assessed the relative amount of SNF2-type ATPases (Brahma, Brg1, SNF2H, SNF2L, CHD3, and CHD5) in three different donor cells as well as in porcine metaphase II oocytes. We performed SCNT with fetal fibroblast cells, olfactory bulb (OB) progenitor cells, and porcine skin originating sphere stem cells (PSOS). We found that OB-NT embryos and PSOS-NT embryos resulted in a higher morulae/blastocysts ratio as compared to fibroblast-NT embryos (23.53%, 16.98%, and 11.63%, respectively; P < 0.05). Fibroblast cells contained a significantly higher amount of SNF2L and CHD3 transcripts while Brg1 and SNF2H were the most expressed transcripts in all the cell lines analyzed. Metaphase II oocyte expression profile appeared to be unique compared to the cell lines analyzed. This work supports our hypothesis that an array of chromatin-remodeling proteins on donor cells may influence the chromatin structure, effect epigenetic reprogramming, and developmental potential.
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Blastocisto/metabolismo , Ensamble y Desensamble de Cromatina , Clonación de Organismos , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/biosíntesis , Técnicas de Transferencia Nuclear , Animales , Blastocisto/patología , Ensamble y Desensamble de Cromatina/genética , Técnicas de Cultivo de Embriones , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica/genética , Metafase/genética , Proteínas Nucleares/genética , Oocitos/metabolismo , Oocitos/patología , PorcinosRESUMEN
This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.
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Estructuras Embrionarias/metabolismo , Lamina Tipo A/metabolismo , Técnicas de Transferencia Nuclear , Porcinos/embriología , Animales , Estructuras Embrionarias/química , Femenino , Lamina Tipo A/análisis , Oocitos/química , Oocitos/metabolismo , Cigoto/química , Cigoto/metabolismoRESUMEN
Porcine xenografts transplanted into primates are rejected in spite of immunosuppression. Identification of the triggering mechanisms and the strategies to overcome them is crucial to achieve long-term graft survival. We hypothesized that porcine CD86 (pCD86) contributes to xenograft rejection by direct activation of host T cells and NK cells. Formerly, we designed the human chimeric molecule hCD152-hCD59 to block pCD86 in cis. To test the efficacy in vivo, we have utilized a pig-to-mouse xenotransplant model. First, we showed that hCD152-hCD59 expression prevents the binding of murine CD28Ig to pCD86 on porcine aortic endothelial cells (PAEC) and dramatically reduces IL-2 secretion by Con A-stimulated mouse splenocytes in coculture. Moreover, IFN-gamma secretion by IL-12-stimulated mouse NK cells was averted after coculture with hCD152-hCD59 PAEC. In vivo, control PAEC implanted under the kidney capsule were rapidly rejected (2-4 weeks) in BALB/c and BALB/c SCID mice. Rejection of hCD152-hCD59 PAEC was significantly delayed in both cases. Signs of immune modulation in the hCD152-hCD59-PAEC BALB/c recipients were identified such as early hyporesponsiveness and diminished antibody response. Thus, simply modifying the donor xenogeneic cell can diminish both T cell and NK cell immune responses. We specifically demonstrate that pCD86 contributes to rejection of porcine xenografts.
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Antígenos CD/sangre , Endotelio Vascular/trasplante , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/sangre , Linfocitos T/inmunología , Trasplante Heterólogo/inmunología , Animales , Antígenos CD/análisis , Antígenos CD/inmunología , Antígenos de Diferenciación/análisis , Aorta , Antígeno B7-2 , Antígenos CD59/análisis , Antígeno CTLA-4 , Técnicas de Cocultivo , Concanavalina A , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Humanos , Interleucina-2/metabolismo , Trasplante de Riñón/inmunología , Trasplante de Riñón/patología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , PorcinosRESUMEN
Olfactory ensheathing cells (OECs) have been shown to mediate remyelination and to stimulate axonal regeneration in a number of in vivo rodent spinal cord studies. However, whether OECs display similar properties in the primate model has not been tested so far. In the present study, we thus transplanted highly-purified OECs isolated from transgenic pigs expressing the alpha1,2 fucosyltransferase gene (H-transferase or HT) gene into a demyelinated lesion of the African green monkey spinal cord. Four weeks posttransplantation, robust remyelination was found in 62.5% of the lesion sites, whereas there was virtually no remyelination in the nontransplanted controls. This together with the immunohistochemical demonstration of the grafted cells within the lesioned area confirmed that remyelination was indeed achieved by OECs. Additional in vitro assays demonstrated 1) that the applied cell suspension consisted of >98% OECs, 2) that the majority of the cells expressed the transgene, and 3) that expression of the HT gene reduced complement activation more than twofold compared with the nontransgenic control. This is the first demonstration that xenotransplantation of characterized OECs into the primate spinal cord results in remyelination.
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Trasplante de Tejido Encefálico , Fucosiltransferasas/metabolismo , Haplorrinos , Vaina de Mielina/metabolismo , Bulbo Olfatorio/trasplante , Regeneración , Médula Espinal/metabolismo , Porcinos , Animales , Animales Modificados Genéticamente , Metabolismo de los Hidratos de Carbono , Trasplante de Células , Células Cultivadas , Proteínas del Sistema Complemento/metabolismo , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Citometría de Flujo , Fucosiltransferasas/genética , Bulbo Olfatorio/citología , Médula Espinal/patología , Trasplante HeterólogoRESUMEN
The field of Regenerative Biology as it applies to Regenerative Medicine is an increasingly expanding area of research with hopes of providing therapeutic treatments for diseases and/or injuries that conventional medicines and even new biologic drug therapies cannot effectively treat. Extensive research in the area of Regenerative Medicine is focused on the development of cells, tissues and organs for the purpose of restoring function through transplantation. The general belief is that replacement, repair and restoration of function is best accomplished by cells, tissues or organs that can perform the appropriate physiologic/metabolic duties better than any mechanical device, recombinant protein therapeutic or chemical compound. Several strategies are currently being investigated and include, cell therapies derived from autologous primary cell isolates, cell therapies derived from established cell lines, cell therapies derived from a variety of stem cells, including bone marrow/mesenchymal stem cells, cord blood stem cells, embryonic stem cells, as well as cells tissues and organs from genetically modified animals. This mini-review is not meant to be exhaustive, but aims to highlight clinical applications for the four areas of research listed above and will address a few key advances and a few of the hurdles yet to be overcome as the technology and science improve the likelihood that Regenerative Medicine will become clinically routine.
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Trasplante de Células , Medicina Regenerativa/tendencias , Ingeniería de Tejidos/tendencias , Animales , Animales Modificados Genéticamente , Trasplante de Médula Ósea , Ensayos Clínicos como Asunto , Predicción , Humanos , Regeneración , Trasplante de Células Madre , Porcinos , Trasplante Autólogo , Trasplante Heterólogo , Trasplante HomólogoRESUMEN
The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.
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Fucosiltransferasas/genética , Galactosiltransferasas/genética , Animales , Antibacterianos/farmacología , Southern Blotting , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Células Cultivadas , Clonación de Organismos , Codón/genética , Cartilla de ADN , Exones/genética , Femenino , Feto/citología , Fibroblastos , Citometría de Flujo , Humanos , Intrones/genética , Masculino , Neomicina/farmacología , Oocitos/fisiología , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , TransfecciónRESUMEN
BACKGROUND: Humoral and cellular defense mechanisms mediate the rejection of transplanted cells, tissues, and organs after allogeneic or xenogeneic transplantation. Inhibition of complement and T-cell costimulation are strategies aimed at increasing transplant survival. METHODS: Engineered novel fusion proteins that contain the functional domains of human CD152 (hCTLA4) or porcine CD152 (pCD152) and human CD59 (hCD152-hCD59, pCD152-hCD59) were developed to form bifunctional chimeric proteins that retain the effector functions of both moieties. Porcine aortic endothelial cells and murine Balb/3T3 cells were transduced or transfected to express the novel fusion proteins. RESULTS: Fluorescence-activated cell sorter analysis of hCD152-hCD59 transduced primary porcine aortic endothelial cells or hCD152-hCD59 and pCD152-hCD59 transfected Balb/3T3 cells determined that the molecules were expressed on the cell surface, and that they retained conformational epitopes. We demonstrate that hCD152-hCD59 and pCD152-hCD59 chimeric proteins inhibit complement-mediated cell lysis. In addition, hCD152-hCD59 or pCD152-hCD59 expression resulted in a significant reduction in T-cell activation as the result of CD152 engagement of porcine CD86 or murine CD80 in when Jurkat cells were cocultured with the hCD152-hCD59 or pCD152-hCD59 expressing cells. Antibody-blocking experiments or phosphatidylinositol phospholipase C removal of the glycosyl-phosphatidylinositol-linked molecules resulted in increased serum-mediated cytolysis and eliminated the costimulatory blockade. CONCLUSIONS: These data illustrate that a single molecule can confer resistance to humoral and cellular immune attack.
Asunto(s)
Formación de Anticuerpos/genética , Antígenos de Diferenciación/inmunología , Antígenos CD59/inmunología , Inmunidad Celular/genética , Inmunoconjugados , Inmunología del Trasplante , Células 3T3 , Abatacept , Animales , Formación de Anticuerpos/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Antígeno B7-2 , Antígenos CD59/genética , Antígenos CD59/metabolismo , Antígeno CTLA-4 , Proteínas del Sistema Complemento/inmunología , Prueba de Complementación Genética , Humanos , Inmunidad Celular/inmunología , Interleucina-2/inmunología , Células Jurkat , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Porcinos , Linfocitos T/inmunologíaRESUMEN
The use of xenogeneic cells or tissues for tissue engineering applications may lead to advances in biomedical research. Hyperacute and delayed rejection are immunologic hurdles that must be addressed to achieve xenograft survival in the pig-to-primate setting. Expression of human alpha1,2-fucosyltransferase (HT) in the donor cell or tissue protects from hyperacute rejection (HAR) by reducing expression of Galalpha1,3-Gal epitope, the major xenoantigen recognized by human natural antibodies. We hypothesized that Galalpha1,3-Gal antigen contributes to delayed tissue rejection. To test this hypothesis, we transplanted control or HT-transgenic engineered porcine cartilage s.c. into alpha1,3-galactosyltransferase knockout (Gal KO) mice. Control porcine cartilage grafted in Gal KO mice was not susceptible to HAR but was rejected in several wk by a prominent cellular immune infiltrate and elevated antibody titers. In contrast, Gal KO mice receiving the HT engineered cartilage showed a markedly reduced anti-pig antibody response and no anti-Galalpha1,3-Gal-elicited antibody response. The HT implants had a mild cellular infiltrate that was confined to the graft periphery. Our study demonstrates that a marked reduction of Galalpha1,3-Gal antigen in HT-transgenic porcine cartilage confers resistance to a process of delayed rejection. Further development of tissue engineering applications that use genetically modified porcine tissues is encouraged.
Asunto(s)
Cartílago/trasplante , Fucosiltransferasas/genética , Rechazo de Injerto/inmunología , Animales , Anticuerpos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Cartílago/inmunología , Cartílago/patología , Células Cultivadas , Condrocitos/inmunología , Disacáridos/análisis , Disacáridos/inmunología , Fucosiltransferasas/metabolismo , Galactosiltransferasas/genética , Expresión Génica , Rechazo de Injerto/patología , Rechazo de Injerto/terapia , Cinética , Ratones , Ratones Noqueados , Modelos Inmunológicos , Monocitos/inmunología , Organismos Modificados Genéticamente , Porcinos , Trasplante Heterólogo , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Manipulation of the pig genome has the potential to improve pig production and offers powerful biomedical applications. Genetic manipulation of mammals has been possible for over two decades, but the technology available has proven both difficult and inefficient. The development of new techniques to enhance efficiency and overcome the complications of random insertion is of importance. Nuclear transfer combined with homologous recombination provides a possible solution: precise genetic modifications in the pig genome may be induced via homologous recombination, and viable offspring can be produced by nuclear transfer using cultured transfected cell lines. The technique is still ineffective, but it is believed to have immense potential. One area that would benefit from the technology is that of xenotransplantation: transgenic pigs are expected to be available as organ donors in the foreseeable future.
Asunto(s)
Transferencia de Embrión , Técnicas Genéticas , Técnicas de Transferencia Nuclear , Animales , Animales Modificados Genéticamente , Línea Celular , Femenino , Masculino , Microinyecciones , Recombinación Genética , Retroviridae/genética , Espermatozoides/patología , Porcinos , Trasplante HeterólogoRESUMEN
Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.