Remarkable progress over the past decade has led to the first clinical studies of stem cell therapy for retinal disease. The unique access retina offers for implantation, monitoring, and ablation is well suited for stem cell trials, and retinal applications have now moved to the forefront of the field of regenerative medicine. Retinal progeny derived from either pluripotent stem cells or tissue-specific retinal and retinal pigment epithelium (RPE) stem cells have the capacity both to replace damaged retina and to provide trophic support that slows disease progression. In contrast, bone marrow and neural stem cells produce nonretinal progeny that provide trophic support but with limited integration and capacity to differentiate into retinal progeny that can replace damaged retinal tissue. Embryonic and induced pluripotent stem cells differentiated into neural retinal and RPE progeny provide an unlimited supply of human cells for transplantation and disease modeling but raise the risks of aberrant differentiation and over proliferation. Tissue-specific stem cells isolated from neural retina or RPE that are naturally committed to retinal fates have a restricted lineage potential that improves the margin of safety. This improved safety of retina and RPE stem cells is balanced, however, by a restricted proliferative potential, which limits the quantity of progeny produced. In this chapter, we review the types of stem cells under development for retinal therapy.
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.
The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress.
The retinal pigment epithelium (RPE) is implicated in many eye diseases, including age-related macular degeneration, and therefore isolating and culturing these cells from recently deceased adult human donors is the ideal source for disease studies. Adult RPE could also be used as a cell source for transplantation therapy for RPE degenerative disease, likely requiring first in vitro expansion of the cells obtained from a patient. Previous protocols have successfully extracted RPE from adult donors; however improvements in yield, cell survival, and functionality are needed. We describe here a protocol optimized for adult human tissue that yields expanded cultures of RPE with morphological, phenotypic, and functional characteristics similar to freshly isolated RPE. These cells can be expanded and cultured for several months without senescence, gross cell death, or undergoing morphological changes. The protocol takes around a month to obtain functional RPE monolayers with accurate morphological characteristics and normal protein expression, as shown through immunohistochemistry analysis, RNA expression profiles via quantitative PCR (qPCR), and transepithelial resistance (TER) measurements. Included in this chapter are steps used to extract RPE from human adult globes, cell culture, cell splitting, cell bleaching, immunohistochemistry, and qPCR for RPE markers, and TER measurements as functional test.
Aging, higher prevalence of diabetes, worsening obesity, and hyperglycemia among potential donors increase the likelihood that pancreata will be declined by transplant centers. Hemoglobin A1c testing, also known as glycated hemoglobin testing, identifies a donors average blood glucose concentration for the preceding 2 to 3 months and is the standard test for identifying prolonged periods of hyperglycemia.
An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.
Retinal degenerative disease has limited therapeutic options and the possibility of stem cell-mediated regenerative treatments is being actively explored for these blinding retinal conditions. The relative accessibility of this central nervous system tissue and the ability to visually monitor changes after transplantation make the retina and adjacent retinal pigment epithelium prime targets for pioneering stem cell therapeutics. Prior work conducted for several decades indicated the promise of cell transplantation for retinal disease, and new strategies that combine these established surgical approaches with stem cell-derived donor cells is ongoing. A variety of tissue-specific and pluripotent-derived donor cells are being advanced to replace lost or damaged retinal cells and/or to slow the disease processes by providing neuroprotective factors, with the ultimate aim of long-term improvement in visual function. Clinical trials are in the early stages, and data on safety and efficacy are widely anticipated. Positive outcomes from these stem cell-based clinical studies would radically change the way that blinding disorders are approached in the clinic.
Since their discovery twenty years ago and prospective isolation a decade later, neural stem cells (NSCs), their progenitors, and differentiated cell derivatives along with other stem-cell based strategies have advanced steadily toward clinical trials, spurred by the immense need to find reparative therapeutics for central nervous system (CNS) diseases and injury. Current phase I/II trials using stem cells in the CNS are the vanguard for the widely anticipated next generation of regenerative therapies and as such are pioneering the stem cell therapy process. While translation has typically been the purview of industry, academic researchers are increasingly driven to bring their findings toward treatments and face challenges in knowledge gap and resource access that are accentuated by the unique financial, manufacturing, scientific, and regulatory aspects of cell therapy. Solutions are envisioned that both address the significant unmet medical need and lead to increased funding for basic and translational research.
Calreticulin is an essential, multifunctional Ca(2+)-binding protein that participates in the regulation of intracellular Ca(2+) homeostasis, cell adhesion, and chaperoning. Calreticulin is abundantly expressed and regulated by androgens in prostate epithelial cells. Given the importance of both calreticulin in multiple essential cellular activities and androgens in prostate cancer, we investigated the possibility of a role for calreticulin in prostate cancer progression. Immunohistochemistry revealed the down-regulation of calreticulin in a subset of human prostate cancer specimens. Prostate cancer cells overexpressing exogenous calreticulin produced fewer colonies in both monolayer culture and soft agar. Furthermore, calreticulin overexpression also inhibited tumor growth in the orthotopic PC3 xenograft tumor model and macroscopic lung metastasis in the rat Dunning AT3.1 prostate tumor model. To address the potential mechanism of calreticulin suppression of prostate cancer, we generated calreticulin mutants with different functional domains deleted. The calreticulin mutants containing the P-domain, which binds to other endoplasmic reticulum chaperone proteins, were sufficient for the suppression of PC3 growth in colony formation assays. Overall, our data support the hypothesis that calreticulin inhibits growth and/or metastasis of prostate cancer cells and that this suppression requires the P-domain.
Vision loss is a major social issue, with more than 20 million people over the age of 18 years affected in the USA alone. Loss of vision is feared more than premature death or cardiovascular disease, according to a recent Society for Consumer Research group survey. The annual direct cost of medical care for the most prevalent eye disease, age-related macular degeneration, was estimated at US$255 billion in 2010 with an additional economic impact of US$88 billion due to lost productivity and the burden of family and community care for visual disability. With the blossoming of human stem cell research, regenerative treatments are now being developed that can help reduce this burden. Positive results from animal studies demonstrate that stem cell-based transplants can preserve and potentially improve vision. This has led to new clinical trials for several eye diseases that are yielding encouraging results. In the next few years, additional trials and longer-term results are anticipated to further develop ocular regenerative therapies, with the potential to revolutionize our approach to ophthalmic disease and damage.
Enhancement of renal allograft function and survival in an era where expanded criteria donors are increasingly used requires validated selection criteria. The goal of this retrospective study was to evaluate the significance of pretransplant donor and allograft parameters to identify risk factors that can be used in a model to predict 1-year allograft outcomes. Donor demographic factors, donor type, and allograft parameters such as biopsy results and machine-measured renal resistance were correlated with 1-year graft outcome. The Kaplan-Meier method was used to estimate graft survival using the categorical predictors of donor type, donor age, and machine measured renal resistance at 1.5, 3, and 5 hours. The log-rank test was used to test the difference in survival curves between cohorts. The Cox regression analysis was used to estimate hazard ratios for machine-measured renal resistance, donor age, donor terminal creatinine level, donors estimated glomerular filtration rate, cold ischemia time, and percent glomerulosclerosis. The data show that machine-measured renal resistance at 3 and 5 hours has a statistically significant inverse relationship to 1-year graft survival. All other risk factors had no correlation with 1-year graft survival. The machine-measured renal resistance at 3 hours is the earliest significant predictor of 1-year allograft outcome.
After spinal cord injury (SCI), loss of cells and damage to ascending and descending tracts can result in paralysis. Current treatments for SCI are based on patient stabilization, and much-needed regenerative therapies are still under development. To activate and instruct stem and progenitor cells or injured tissue to aid SCI repair, it is important to modify the injury environment for a protracted period, to allow time for cell activation, proliferation and appropriate fate differentiation. Shh plays a critical role in spinal cord formation, being involved in multiple processes: it promotes production of motor neurons and oligodendrocytes from ventral cord progenitor cells and serves as an axon guidance molecule. Hence Shh is a candidate pleiotropic beneficial environmental factor for spinal cord regeneration. Here we show that administration of biodegradable microspheres that provide sustained, controlled delivery of Shh resulted in significant functional improvement in two different mouse models of SCI: contusion and dorsal hemioversection. The mechanism is multifactorial, involving increased proliferation of endogenous NG2+ oligodendrocyte lineage cells, decreased astrocytic scar formation and increased sprouting and growth of corticospinal (CST) and raphespinal tract (RST) fibers. Thus, long-term administration of Shh is a potential valuable therapeutic intervention for SCI.
The retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. It begins as a plastic tissue, capable, in some species, of generating lens and retina, but differentiates early in development and remains normally nonproliferative throughout life. Here we show that a subpopulation of adult human RPE cells can be activated in vitro to a self-renewing cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers, proliferates extensively, and can redifferentiate into stable cobblestone RPE monolayers. Clonal studies demonstrate that RPESCs are multipotent and in defined conditions can generate both neural and mesenchymal progeny. This plasticity may explain human pathologies in which mesenchymal fates are seen in the eye, for example in proliferative vitroretinopathy (PVR) and phthisis bulbi. This study establishes the RPESC as an accessible, human CNS-derived multipotent stem cell, useful for the study of fate choice, replacement therapy, and disease modeling.
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