Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.
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ABSTRACT:
SUBMITTER: Liu Z
PROVIDER: S-EPMC7878718 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeWe tested what native features have been preserved with a new culture protocol for adult human RPE.MethodsWe cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used.ResultsConfluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 ?·cm2 (mean ± SEM, n = 31). Application of 100 ?M adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 ?L·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01).ConclusionsThe new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.
| S-EPMC4640474 | biostudies-literature
Project description:Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies.
| S-EPMC3916756 | biostudies-literature
Project description:Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.
| S-EPMC6329375 | biostudies-literature
Project description:BackgroundHuman pluripotent stem cells (hPSCs) provide a promising cell source for retinal cell replacement therapy but often lack standardized cell production and live-cell shipment logistics as well as rigorous analyses of surgical procedures for cell transplantation in the delicate macula area. We have previously established a xeno- and feeder cell-free production system for hPSC differentiated retinal pigment epithelial (RPE) cells, and herein, a novel immunosuppressed non-human primate (NHP) model with a disrupted ocular immune privilege is presented for transplanting human embryonic stem cell (hESC)-derived RPE on a scaffold, and the safety and submacular graft integration are assessed. Furthermore, the feasibility of intercontinental shipment of live hESC-RPE is examined.MethodsCynomolgus monkeys were systemically immunosuppressed and implanted with a hESC-RPE monolayer on a permeable polyester-terephthalate (PET) scaffold. Microscope-integrated intraoperative optical coherence tomography (miOCT)-guided surgery, postoperative follow-up incorporated scanning laser ophthalmoscopy, spectral domain (SD-) OCT, and full-field electroretinography (ERG) were used as outcome measures. In addition, histology was performed after a 28-day follow-up.ResultsIntercontinental cell shipment, which took >30 h from the manufacturing to the transplantation site, did not alter the hESC-RPE quality. The submacular hESC-RPE xenotransplantation was performed in 11 macaques. The miOCT typically revealed foveal disruption. ERG showed amplitude and peak time preservation in cases with favorable surgical outcomes. Histology confirmed photoreceptor preservation above the grafts and in vivo phagocytosis by hESC-RPE, albeit evidence of cytoplasmic redistribution of opsin in photoreceptors and glia hypertrophy. The immunosuppression protocol efficiently suppressed retinal T cell infiltration and microglia activation.ConclusionThese results suggest both structural and functional submacular integrations of hESC-RPE xenografts. It is anticipated that surgical technique refinement will further improve the engraftment of macular cell therapeutics with significant translational relevance to improve future clinical trials.
| S-EPMC8314642 | biostudies-literature
Project description:Islet transplantation effectively treats diabetes but relies on immune suppression and is practically limited by the number of cadaveric islets available. An alternative cellular source is insulin-producing cells derived from pluripotent cell sources. Three animal cohorts were used in the current study to evaluate whether an oxygen-providing macro-encapsulation device, '?AIR', could function in conjunction with human embryonic stem cells (hESCs) and their derivatives. The first cohort received macro-encapsulated undifferentiated hESCs, a second cohort received hESCs differentiated to a pancreatic progenitor state with limited endocrine differentiation. A reference cohort received human islets. Macro-encapsulation devices were implanted subcutaneously and monitored for up to 4 months. Undifferentiated pluripotent stem cells did not form teratoma but underwent cell death following implantation. Human C-peptide (hC- peptide) was detectable in host serum one week after implantation for both other cohorts. hC-peptide levels decreasing over time but remained detectable up to the end of the study. Key factors associated with mature endocrine cells were observed in grafts recovered from cohorts containing islets and hESC-derivatives including C-peptide, insulin, glucagon and urocortin 3. We conclude that the '?AIR' macroencapsulation device is compatible with both human islets and pluripotent derivatives, but has a limited capability of sustaining undifferentiated pluripotent cells.
| S-EPMC6816271 | biostudies-literature
Project description:PurposeRPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems.MethodsEMT of human stem cell-derived RPE monolayers was induced by either enzymatic dissociation or modulation of TGF-β signaling. Transcriptomic analysis of cells at different stages of EMT was performed by RNA-sequencing, and select findings were confirmed by reverse transcription quantitative PCR and immunostaining. An ingenuity pathway analysis (IPA) was performed to identify signaling pathways and regulatory networks associated with EMT.ResultsProteocollagenolytic enzymatic dissociation and cotreatment with TGF-β and TNF-α both induce EMT in human stem cell-derived RPE monolayers, leading to an increased expression of mesenchymal factors and a decreased expression of RPE differentiation-associated factors. Ingenuity pathway analysis identified the upstream regulators of the RPE-EMT regulatory networks and identified master switches and nodes during RPE-EMT. Of particular interest was the identification of widespread dysregulation of axon guidance molecules during RPE-EMT progression.ConclusionsThe temporal transcriptome profiles described here provide a comprehensive resource of the dynamic signaling events and the associated biological pathways that underlie RPE-EMT onset. The pathways defined by these studies may help to identify targets for the development of novel therapeutic targets for the treatment of retinal disease.
| S-EPMC8024778 | biostudies-literature
Project description:Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient's blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.
| S-EPMC7408794 | biostudies-literature
Project description:Adeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this work, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression.Three female Macaca fascicularis were intravenously injected with 1 x 10(13) genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5 x 10(12) genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression.Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system.These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.
| S-EPMC3412719 | biostudies-literature
Project description:The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-? mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.
| S-EPMC5298941 | biostudies-literature
Project description:BACKGROUND:Primate neurobiologists use chronically implanted devices such as pedestals for head stabilization and chambers to gain access to the brain and study its activity. Such implants are skull-mounted, and made from a hard, durable material, such as titanium. NEW METHOD:Here, we present a low-cost method of creating customized 3D-printed cranial implants that are tailored to the anatomy of individual animals. We performed pre-surgical computed tomography (CT) and magnetic resonance (MR) scans to generate three-dimensional (3D) models of the skull and brain. We then used 3D modelling software to design implantable head posts, chambers, and a pedestal anchorage base, as well as craniotomy guides to aid us during surgery. Prototypes were made from plastic or resin, while implants were 3D-printed in titanium. The implants underwent post-processing and received a coating of osteocompatible material to promote bone integration. RESULTS:Their tailored fit greatly facilitated surgical implantation, and eliminated the gap between the implant and the bone. To date, our implants remain robust and well-integrated with the skull. COMPARISON WITH EXISTING METHOD(S):Commercial-off-the-shelf solutions typically come with a uniform, flat base, preventing them from sitting flush against the curved surface of the skull. This leaves gaps for fluid and tissue ingress, increasing the risk of microbial infection and tissue inflammation, as well as implant loss. CONCLUSIONS:The use of 3D printing technology enabled us to quickly and affordably create unique, complex designs, avoiding the constraints levied by traditional production methods, thereby boosting experimental success and improving the wellbeing of the animals.
| S-EPMC5482398 | biostudies-literature