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Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

ABSTRACT: PURPOSE:Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. METHODS:Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. RESULTS:OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. CONCLUSIONS:OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. TRANSLATIONAL RELEVANCE:Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

SUBMITTER: McGill TJ

PROVIDER: S-EPMC5472365 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

McGill Trevor J TJ Bohana-Kashtan Osnat O Stoddard Jonathan W JW Andrews Michael D MD Pandit Neelay N Rosenberg-Belmaker Lior R LR Wiser Ofer O Matzrafi Limor L Banin Eyal E Reubinoff Benjamin B Netzer Nir N Irving Charles C

Translational vision science & technology 20170614 3

<h4>Purpose</h4>Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats.<h4>Methods</h4>Postnatal (P) ...[more]

PMID: 28626601

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Project description:BackgroundHuman mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines.MethodsIn the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium).ResultsThe results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum.ConclusionsThe findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.

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.

Project description:BackgroundAge-related macular degeneration (AMD) is the leading cause of blindness in the elderly due in large part to age-dependent atrophy of retinal pigment epithelium (RPE) cells. RPE cells form a monolayer located between the choroid and the outer segments of photoreceptors, playing multifarious roles in maintenance of visual function. Allogeneically induced pluripotent stem cell-derived RPE (iPSC-RPE or iRPE) has become a potential approach for providing an abundant source of donors for clinical cell products. Transplantation of iRPE has been proven effective in rescuing impaired retinas in Royal College of Surgeons (RCS) rats after approximately 5 to 6 weeks. Here, we explore the long-term (19 weeks) safety and efficacy of human iRPE cell transplantation in pre-clinical animal models.MethodsThe expression of human RPE-specific markers in iRPE cells was determined using immunofluorescence staining. For the proliferative test, Ki-67 expression was also verified by immunofluorescence and flow cytometric analysis. Then, iRPE cells were transplanted into the subretinal space of immune-deficient NOD/SCID/IL-2Rgcnull (NSG) mice to assess their safety. To evaluate whether the transplanted cells could survive and rescue visual function, we performed color fundus photography, focal electroretinogram and immunostaining after delivering iRPE cells into the subretinal space of RCS rats.ResultsHuman iRPE cells expressed native RPE-specific markers, such as microphthalmia-associated transcription factor (MiTF), retinal pigment epithelium-specific 65-kDa protein (RPE65) and tight-junction associated structural protein (ZO-1), and their proliferative capacity (Ki-67 expression) was poor after 25 days of induction. A tumorigenicity test revealed no tumor formation or abnormal proliferation in the immunodeficient mice after subretinal injection of 5×105 iRPE cells. The transplanted iRPE cells survived for at least 19 weeks and maintained visual function for 15 weeks.ConclusionsIn the present study, we provided further evidence for the use of human iRPE transplantation to treat retinal degenerative disease in pre-clinical animal models. Therefore, we consider human iRPE cells a promising source of cell replacement therapy for AMD.

Project description:BACKGROUND:Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). METHODS:hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. RESULTS:HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. CONCLUSIONS:Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

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Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

Publications

Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

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