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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Article
  • Peer Reviewed
UC Santa Barbara Previously Published Works (2016)
Age-related macular degeneration (AMD) is the leading cause of blindness in the western world, which severely decreases the quality of life in the patients and places an economic burden on their families and society. The disease is caused by the dysfunction of a specialized cell layer in the back of the eye called the retinal pigmented epithelium (RPE). Pluripotent stem cells can provide an unlimited source of RPE, and laboratories around the world are investigating their potential as therapies for AMD. To ensure the precise delivery of functional RPE to the diseased site, some groups are developing a therapy composed of mature RPE monolayers on a supportive scaffold for transplantation as an alternative to injecting a single-cell suspension. This review summarizes methods of generating RPE from pluripotent stem cells, compares biodegradable and biostable materials as scaffolds, and describes the specific combination of human embryonic stem cell-derived RPE on Parylene-C membranes, which is scheduled to begin clinical trials in the United Sates in 2016. Stem cell-derived RPE monolayers on scaffolds hold great promise for the treatment of AMD and other retinal diseases.
    Cover page: Pluripotent Stem Cell-Based Therapies in Combination with Substrate for the Treatment of Age-Related Macular Degeneration
    • Thesis
    • Peer Reviewed
    UC Santa Barbara Electronic Theses and Dissertations (2014)

    Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly and is characterized by the death of the retinal pigmented epithelium (RPE), the cell layer located behind the retina. The RPE maintains the health of the primary cells responsible for vision, the photoreceptors. As AMD progresses, the RPE degrades, which causes the death of the photoreceptors and a debilitating loss of sight. Human embryonic stem cells (hESCs) can generate a limitless source of RPE for cellular therapies, therefore efforts to derive RPE from human embryonic stem cells (hESCs) to graft into AMD patients are under development. This thesis addresses two of the obstacles that hinder the production of these therapeutical cells. First, to manufacture cells for clinical use, it is desirable for procedures to be performed under defined conditions free of non-human reagents (xeno-free). Therefore, a novel, biomimetic, xeno-free RGD-containing copolymer designed for cell culture was investigated and found to support healthy hESC cultures and permit their differentiation into functional RPE. These stem cells and hESC-RPE demonstrate similar gene expression, protein localization and phagocytic function as cultures grown on a xenogeneic substrate. The second obstacle pertains to the intense time requirement needed to differentiate hESCs into RPE. Therefore, we recapitulated the signaling events during early embryonic development to expedite the production of hESC-RPE from 1-3 months to 14 days. However, this protocol employs full length recombinant growth factors and animal derivatives. These components represent a challenge in employing these cells as therapies due to the risk of exposure to non-human immunogens. Preliminary work to replace the xenogeneic substrate and recombinant growth factors with small molecules is presented. These pilot studies demonstrate that a xeno-free substrate and dual-Smad inhibition via small molecules can rapidly and efficiently differentiate hESCs into a population of cells expressing a pigmentation marker. These studies contribute to the development of defined, xeno-free methods for cellular manufacture which can further the translation of stem cell therapies to the clinic.

      Cover page: Xeno-Free Derivation of Retinal Pigmented Epithelium from Human Pluripotent Stem Cells
      • Article
      • Peer Reviewed
      UC Santa Barbara Previously Published Works (2014)
      We developed an interactive exercise to teach students how to draw the structures of the 20 standard amino acids and to identify the one-letter abbreviations by modifying the familiar game of "Hangman." Amino acid structures were used to represent single letters throughout the game. To provide additional practice in identifying structures, hints to the answers were written in "amino acid sentences" for the students to translate. Students were required to draw the structure of the corresponding letter they wished to guess on a whiteboard. Each student received a reference sheet of the structures and abbreviations, but was required to draw from memory when guessing a letter. Preassessments and postassessments revealed a drastic improvement in the students' ability to recognize and draw structures from memory. This activity provides a fun, educational game to play in biochemistry discussion sections or during long incubations in biochemistry laboratories.
        Cover page: Interactive Hangman teaches amino acid structures and abbreviations
        • Article
        • Peer Reviewed
        UC Santa Barbara Previously Published Works (2015)
        Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies.
          Cover page: Defined Culture of Human Embryonic Stem Cells and Xeno‐Free Derivation of Retinal Pigmented Epithelial Cells on a Novel, Synthetic Substrate
          • Article
          • Peer Reviewed
          UC Santa Barbara Previously Published Works (2022)
          Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available, multiple clinical trials investigating retinal pigmented epithelial cells derived from human pluripotent stem cells (hPSC-RPE) as a cellular replacement therapeutic are currently underway. It has been estimated that a production capacity of >109 RPE cells annually would be required to treat the afflicted population, but current manufacturing protocols are limited, being labor-intensive and time-consuming. Microcarrier technology has enabled high-density propagation of many adherent mammalian cell types via monolayer culture on surfaces of uM-diameter matrix spheres; however, few studies have explored microcarrier-based culture of RPE cells. Here, we provide an approach to the growth, maturation, and differentiation of hPSC-RPE cells on Cytodex 1 (C1) and Cytodex 3 (C3) microcarriers. We demonstrate that hPSC-RPE cells adhere to microcarriers coated with Matrigel, vitronectin or collagen, and mature in vitro to exhibit characteristic epithelial cell morphology and pigmentation. Microcarrier-grown hPSC-RPE cells (mcRPE) are viable; metabolically active; express RPE signature genes including BEST1, RPE65, TYRP1, and PMEL17; secrete the trophic factors PEDF and VEGF; and demonstrate phagocytosis of photoreceptor outer segments. Furthermore, we show that undifferentiated hESCs also adhere to Matrigel-coated microcarriers and are amenable to directed RPE differentiation. The capacity to support hPSC-RPE cell cultures using microcarriers enables efficient large-scale production of therapeutic RPE cells sufficient to meet the treatment demands of a large AMD patient population.
            Cover page: Microcarrier-Based Culture of Human Pluripotent Stem-Cell-Derived Retinal Pigmented Epithelium.
            • Article
            • Peer Reviewed
            UC Santa Barbara Previously Published Works (2016)

            Purpose

            The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE.

            Methods

            This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function.

            Results

            We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE.

            Conclusions

            The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.
              Cover page: Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods
              • Article
              • Peer Reviewed
              UC Santa Barbara Previously Published Works (2021)
              Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.
                Cover page: Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo.
                • Article
                • Peer Reviewed
                UC Santa Barbara Previously Published Works (2022)
                Cell-based therapies face challenges, including poor cell survival, immune rejection, and integration into pathologic tissue. We conducted an open-label phase 1/2a clinical trial to assess the safety and preliminary efficacy of a subretinal implant consisting of a polarized monolayer of allogeneic human embryonic stem cell-derived retinal pigmented epithelium (RPE) cells in subjects with geographic atrophy (GA) secondary to dry age-related macular degeneration. Postmortem histology from one subject with very advanced disease shows the presence of donor RPE cells 2 years after implantation by immunoreactivity for RPE65 and donor-specific human leukocyte antigen (HLA) class I molecules. Markers of RPE cell polarity and phagocytosis suggest donor RPE function. Further histologic examination demonstrated CD34+ structures beneath the implant and CD4+, CD68+, and FoxP3+ cells in the tissue. Despite significant donor-host HLA mismatch, no clinical signs of retinitis, vitreitis, vasculitis, choroiditis, or serologic immune response were detected in the deceased subject or any other subject in the study. Subretinally implanted, HLA-mismatched donor RPE cells survive, express functional markers, and do not elicit clinically detectable intraocular inflammation or serologic immune responses even without long-term immunosuppression.
                  Cover page: Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration.
                  • Article
                  • Peer Reviewed
                  UC Santa Barbara Previously Published Works (2021)
                  PURPOSE: To report 1-year follow-up of a phase 1/2a clinical trial testing a composite subretinal implant having polarized human embryonic stem cell (hESC)-derived retinal pigment epithelium (RPE) cells on an ultrathin parylene substrate in subjects with advanced non-neovascular age-related macular degeneration (NNAMD). METHODS: The phase 1/2a clinical trial included 16 subjects in two cohorts. The main endpoint was safety assessed at 365 days using ophthalmic and systemic exams. Pseudophakic subjects with geographic atrophy (GA) and severe vision loss were eligible. Low-dose tacrolimus immunosuppression was utilized for 68 days in the peri-implantation period. The implant was delivered to the worst seeing eye with a custom subretinal insertion device in an outpatient setting. A data safety monitoring committee reviewed all results. RESULTS: The treated eyes of all subjects were legally blind with a baseline best-corrected visual acuity (BCVA) of ≤ 20/200. There were no unexpected serious adverse events. Four subjects in cohort 1 had serious ocular adverse events, including retinal hemorrhage, edema, focal retinal detachment, or RPE detachment, which was mitigated in cohort 2 using improved hemostasis during surgery. Although this study was not powered to assess efficacy, treated eyes from four subjects showed an increased BCVA of >5 letters (6-13 letters). A larger proportion of treated eyes experienced a >5-letter gain when compared with the untreated eye (27% vs. 7%; P = not significant) and a larger proportion of nonimplanted eyes demonstrated a >5-letter loss (47% vs. 33%; P = not significant). CONCLUSIONS: Outpatient delivery of the implant can be performed routinely. At 1 year, the implant is safe and well tolerated in subjects with advanced dry AMD. TRANSLATIONAL RELEVANCE: This work describes the first clinical trial, to our knowledge, of a novel implant for advanced dry AMD.
                    Cover page: One-Year Follow-Up in a Phase 1/2a Clinical Trial of an Allogeneic RPE Cell Bioengineered Implant for Advanced Dry Age-Related Macular Degeneration.
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