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Although the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.

Autophagy dysfunction has been associated with several neurodegenerative diseases including glaucoma, characterized by the degeneration of retinal ganglion cells (RGCs). However, the mechanisms by which autophagy dysfunction promotes RGC damage remain unclear. Here, we hypothesized that perturbation of the autophagy pathway results in increased autophagic demand, thereby downregulating signaling through mammalian target of rapamycin complex 1 (mTORC1), a negative regulator of autophagy, contributing to the degeneration of RGCs. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor adenosine monophosphate-activated protein kinase (AMPK), along with subsequent neurodegeneration in RGCs differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated variant of Optineurin (OPTN-E50K). Similarly, the microbead occlusion model of glaucoma resulting in ocular hypertension also exhibited autophagy disruption and mTORC1 downregulation. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN-E50K RGCs. Taken together, these results highlight an important balance between autophagy and mTORC1 signaling essential for RGC homeostasis, while disruption to these pathways contributes to neurodegenerative features in glaucoma, providing a potential therapeutic target to prevent neurodegeneration.

Neuronal repopulation achieved through transplantation or transdifferentiation from endogenous sources holds tremendous potential for restoring function in chronic neurodegenerative disease or acute injury. Key to the evaluation of neuronal engraftment is the definitive discrimination of new or donor neurons from preexisting cells within the host tissue. Recent work has identified mechanisms by which genetically encoded donor cell reporters can be transferred to host neurons through intercellular material transfer. In addition, labeling transplanted and endogenously transdifferentiated neurons through viral vector transduction can yield misexpression in host cells in some circumstances. These issues can confound the tracking and evaluation of repopulated neurons in regenerative experimental paradigms. Using the retina as an example, we discuss common reasons for artifactual labeling of endogenous host neurons with donor cell reporters and suggest strategies to prevent erroneous conclusions based on misidentification of cell origin.

Mitochondrial dysfunctions are widely afflicted in central nervous system (CNS) disorders with minimal understanding on how to improve mitochondrial homeostasis to promote neuroprotection. Here we used human stem cell differentiated retinal ganglion cells (hRGCs) of the CNS, which are highly sensitive towards mitochondrial dysfunctions due to their unique structure and function, to identify mechanisms for improving mitochondrial quality control (MQC). We found that hRGCs are efficient in maintaining mitochondrial homeostasis through rapid degradation and biogenesis of mitochondria under acute damage. Using a glaucomatous Optineurin mutant (E50K) stem cell lines, we saw that at basal level mutant hRGCs possess less mitochondrial mass and suffer mitochondrial swelling due to excess ATP production load. Activation of mitochondrial biogenesis through pharmacological inhibition of the Tank binding kinase 1 (TBK1) restored energy homeostasis, mitigated mitochondrial swelling with neuroprotection against acute mitochondrial damage for glaucomatousE50KhRGCs, revealing a novel neuroprotection mechanism.

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughputin vivoscreening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENTHere, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCsin vivo. To our knowledge, this is the first report demonstratingin vivoneuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

Summary Retinal ganglion cells (RGCs) form the connection between the eye and the brain, with this connectivity disrupted in numerous blinding disorders. Previous studies have demonstrated the ability to derive RGCs from human pluripotent stem cells (hPSCs); however, these cells exhibited some characteristics that indicated a limited state of maturation. Among the many factors known to influence RGC development in the retina, astrocytes are known to play a significant role in their functional maturation. Thus, efforts of the current study examined the functional maturation of hPSC-derived RGCs, including the ability of astrocytes to modulate this developmental timeline. Morphological and functional properties of RGCs were found to increase over time, with astrocytes significantly accelerating the functional maturation of hPSC-derived RGCs. The results of this study clearly demonstrate the functional and morphological maturation of RGCs in vitro, including the effects of astrocytes on the maturation of hPSC-derived RGCs., Highlights • Improved maturation of hPSC-derived RGCs in a temporally appropriate manner • Co-cultures of RGCs and astrocytes recapitulate cellular interactions in the retina • Astrocytes enhance functional and morphological maturation of hPSC-derived RGCs, In this article, VanderWall and colleagues demonstrate the morphological and functional maturation of hPSC-derived RGCs over time, including the role of astrocytes in their functional maturation. Results indicated that hPSC-derived RGCs were capable of exhibiting robust neurite outgrowth and functional properties, with the direct contact with astrocytes significantly enhancing this maturation. As astrocytes are a major component of the nerve fiber layer and optic nerve, the results of these studies constitute a model for studying these cellular interactions in vitro.

Summary The development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway., Graphical abstract, Highlights • Human stem cell-derived RGC axons respond to target-derived cues • Assembloids were generated between retinal, thalamic, and cortical organoids • Retinofugal projections robustly extend toward thalamic targets, In this article, Fligor and colleagues present the development of a three-dimensional assembloid model for retinofugal projections, comprising retinal, thalamic, and cortical organoids. Results presented include a detailed analysis of enhanced RGC survival and robust axonal outgrowth and provide the opportunity to explore methods for RGC regeneration and disease modeling.

Humans have limited ability to repair damaged central nervous system neurons, such as retinal ganglion cells, making it critical to advance towards ways to reconstruct a patient’s retina if one suffers from trauma or a neurodegenerative disease of the eye, such as glaucoma and other blinding disorders. The use of human pluripotent stem cells (hPSCs) provides powerful approaches to the development of novel therapeutic strategies to blinding diseases due to their ability to self-renew and form any cell type in the human body through targeted differentiation. Recently, hPSCs have been shown to self-form retinal organoids, which are three-dimensional aggregates of cells that mimic the spatial and temporal organization of the human retina in vitro. Therefore, retinal organoids provide an in vitro model of the human retina for drug screening, disease modeling, and cell replacement therapies. However, before these approaches can be fully realized, a critical need exists to refine these differentiation methods as current protocols lead to heterogeneity of organoids produced, as well as inconsistency in their shape and size, leading to significant variability within experiments. Thus, the goal of this research is to optimize current retinal organoid differentiation protocols to generate highly reproducible retinal organoids that are consistent in size, shape and cellular composition across various stem cell lines that can be used for disease modeling and high-throughput applications. The size and shape of retinal organoids was experimentally controlled for through the use of low-adhesion 96 well U-bottom plates with centrifugation to induce quick reaggregation of individual undifferentiated hPSCs. These aggregates were compared to those generated through traditional methods in which whole colonies were mechanically lifted. Resulting retinal organoids at early and later stages showed greater reproducibility in size and shape across different stem cell lines, demonstrating greater suitability for direct comparisons in disease modeling and drug screening experiments.

Retinal ganglion cells (RGCs) are a heterogeneous population of neurons, comprised of numerous subtypes that work synchronously to transmit visual information to the brain. In blinding disorders such as glaucoma, RGCs are the main cell type to degenerate and lead to loss of vision. Previous studies have identified and characterized a variety of RGC subtypes in animal models, although only a handful of studies demonstrate the differential loss of these RGC subtypes in response to disease or injury. Thus, efforts of the current study utilized both chronic (bead occlusion) and acute (optic nerve crush, ONC) rat models to characterize disease response and differential loss of RGC subtypes. Bead occlusion and ONC retinas demonstrated significant RGC loss, glial reactivity and apoptosis compared to control retinas. Importantly, bead occlusion and ONC retinas resulted in differential subtype-specific loss of RGCs, with a high susceptibility for alpha- and direction selective-RGCs and preferential survival of ipRGCs. Results of this study serve as an important foundation for future experiments focused on the mechanisms resulting in the loss of RGCs in optic neuropathies, as well as the development of targeted therapeutics for RGC subtype-specific neuroprotection.

Human pluripotent stem cells (hPSCs) possess the remarkable ability to differentiate into any cell type of the body, including those of the retina. Through the differentiation of these cells as retinal organoids, it is now possible to model the spatial and temporal development of the human retina using hPSCs, in which retinal progenitor cells produce the entire repertoire of retinal cells, first differentiating into retinal ganglion cells and ending with mature photoreceptors, bipolar cells, and Müller glia. Importantly, retinal organoids self-assemble into laminated structures that recapitulate the layering of the human retina with a retinal ganglion cell layer lining the inner layer and a distinctly separate photoreceptor layer occupying the outer layers. This organoid technology has provided access to human tissue for developmental and disease modeling, as well as translational applications such as high throughput drug screening and cell replacement therapies. However, the differentiation of retinal organoids does require some expertise and multiple strategies produce inconsistent results. Here, we describe in detail a well-established and relatively simple method for the generation of retinal organoids.

Human pluripotent stem cells (hPSCs) possess the remarkable ability to differentiate into any cell type of the body, including those of the retina. Through the differentiation of these cells as retinal organoids, it is now possible to model the spatial and temporal development of the human retina using hPSCs, in which retinal progenitor cells produce the entire repertoire of retinal cells, first differentiating into retinal ganglion cells and ending with mature photoreceptors, bipolar cells, and Muller glia. Importantly, retinal organoids self-assemble into laminated structures that recapitulate the layering of the human retina with a retinal ganglion cell layer lining the inner layer and a distinctly separate photoreceptor layer occupying the outer layers. This organoid technology has provided access to human tissue for developmental and disease modeling, as well as translational applications such as high throughput drug screening and cell replacement therapies. However, the differentiation of retinal organoids does require some expertise and multiple strategies produce inconsistent results. Here, we describe in detail a well-established and relatively simple method for the generation of retinal organoids.

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

Glaucoma, one of the most common causes of blindness in developing countries today, involves a progressive loss of neural cells in the optic nerve that leads to progressive, irreversible vision loss. Increased intraocular pressure (IOP) presents as a major risk factor for glaucoma, although there exist cases of glaucoma patients with normal IOP that exhibit damage to retinal ganglion cells (RGCs) and the optic nerve. However, treatment approaches have maintained their focus on modifying IOP due to a lack of other modifiable risks factors. Traditional concepts in glaucoma involve the neuronal environment and external effects as a source of causative factors; however, studies have yet to investigate whether the molecular profile of RGCs in glaucoma patients makes them more vulnerable and/or susceptible to external damage. Our hypothesis states that molecular changes at the whole cell, gene expression, and electrophysiological level of the neurons can contribute to their degeneration. Herein, we briefly describe different types of glaucoma and any similarities to different molecular and cellular features of neurodegeneration. To test our hypothesis, we describe human induced pluripotent stem cells (hiPSCs) as a reliable cellular tool to model neurodegenerative aspects of glaucoma to reveal the multiple pathological molecular mechanisms underlying disease development.

SummaryRetinal ganglion cells (RGCs) serve as the primary connection between the eye and the brain, with this connection disrupted in glaucoma. Numerous cellular mechanisms have been associated with glaucomatous neurodegeneration, and useful models of glaucoma allow for the precise analysis of degenerative phenotypes. Human pluripotent stem cells (hPSCs) serve as powerful tools for studying human neurodegenerative diseases, particularly cellular mechanisms underlying degeneration. Thus, efforts were initially focused upon the use of hPSCs with an E50K mutation in the Optineurin (OPTN) gene. CRISPR/Cas9 gene editing was used to introduce the OPTN(E50K) mutation into existing lines of hPSCs, as well as the generation of isogenic control lines from OPTN(E50K) patient-derived hPSC lines. OPTN(E50K) RGCs exhibited numerous neurodegenerative deficits, including neurite retraction, autophagy dysfunction, apoptosis, and increased excitability. The results of this study provide an extensive analysis of the OPTN(E50K) mutation in hPSC-derived RGCs, with the opportunity to develop novel treatments for glaucoma.

Summary Retinal ganglion cells (RGCs) serve as the connection between the eye and the brain, with this connection disrupted in glaucoma. Numerous cellular mechanisms have been associated with glaucomatous neurodegeneration, and useful cellular models of glaucoma allow for the precise analysis of degenerative phenotypes. Human pluripotent stem cells (hPSCs) serve as powerful tools for studying human disease, particularly cellular mechanisms underlying neurodegeneration. Thus, efforts focused upon hPSCs with an E50K mutation in the Optineurin (OPTN) gene, a leading cause of inherited forms of glaucoma. CRISPR/Cas9 gene editing introduced the OPTN(E50K) mutation into existing lines of hPSCs, as well as generating isogenic controls from patient-derived lines. RGCs differentiated from OPTN(E50K) hPSCs exhibited numerous neurodegenerative deficits, including neurite retraction, autophagy dysfunction, apoptosis, and increased excitability. These results demonstrate the utility of OPTN(E50K) RGCs as an in vitro model of neurodegeneration, with the opportunity to develop novel therapeutic approaches for glaucoma., Graphical Abstract, Highlights • CRISPR/Cas9 engineering of OPTN(E50K) glaucomatous hPSCs and isogenic controls • RNA sequencing displays important pathways linked to glaucomatous neurodegeneration • OPTN(E50K) RGCs encompass numerous neurodegenerative mechanisms, In this article, VanderWall, Huang and colleagues present the extensive characterization of the OPTN(E50K) mutation in hPSC-derived RGCs using CRISPR/Cas9-engineered disease lines and isogenic controls. Results presented a detailed analysis of numerous neurodegenerative phenotypes observed in OPTN(E50K) RGCs and provide the opportunity to explore new targets for therapeutic intervention.

In the present study, we investigated whether mutant huntingtin (mHTT) impairs mitochondrial functions in human striatal neurons derived from induced pluripotent stem cells (iPSCs). Striatal neurons and astrocytes derived from iPSCs from unaffected individuals (Ctrl) and Huntington’s disease (HD) patients with HTT gene containing increased number of CAG repeats were used to assess the effect of mHTT on bioenergetics and mitochondrial superoxide anion production. The human neurons were thoroughly characterized and shown to express MAP2, DARPP32, GABA, synapsin, and PSD95. In human neurons and astrocytes expressing mHTT, the ratio of mHTT to wild-type huntingtin (HTT) was 1:1. The human neurons were excitable and could generate action potentials, confirming successful conversion of iPSCs into functional neurons. The neurons and astrocytes from Ctrl individuals and HD patients had similar levels of ADP and ATP and comparable respiratory and glycolytic activities. The mitochondrial mass, mitochondrial membrane potential, and superoxide anion production in human neurons appeared to be similar regardless of mHTT presence. The present results are in line with the results obtained in our previous studies with isolated brain mitochondria and cultured striatal neurons from YAC128 and R6/2 mice, in which we demonstrated that mutant huntingtin at early stages of HD pathology does not deteriorate mitochondrial functions. Overall, our results argue against bioenergetic deficits as a factor in HD pathogenesis and suggest that other detrimental processes might be more relevant to the development of HD pathology.

Human pluripotent stem cell (hPSC) technology has revolutionized the field of biology through the unprecedented ability to study the differentiation of human cells in vitro. In the past decade, hPSCs have been applied to study development, model disease, develop drugs, and devise cell replacement therapies for numerous biological systems. Of particular interest is the application of this technology to study and treat optic neuropathies such as glaucoma. Retinal ganglion cells (RGCs) are the primary cell type affected in these diseases, and once lost, they are unable to regenerate in adulthood. This necessitates the development of strategies to study the mechanisms of degeneration as well as develop translational therapeutic approaches to treat early- and late-stage disease progression. Numerous protocols have been established to derive RGCs from hPSCs, with the ability to generate large populations of human RGCs for translational applications. In this review, the key applications of hPSCs within the retinal field are described, including the use of these cells as developmental models, disease models, drug development, and finally, cell replacement therapies. In greater detail, the current report focuses on the differentiation of hPSC-derived RGCs and the many unique characteristics associated with these cells in vitro including their genetic identifiers, their electrophysiological activity, and their morphological maturation. Also described is the current progress in the use of patient-specific hPSCs to study optic neuropathies affecting RGCs, with emphasis on the use of these RGCs for studying disease mechanisms and pathogenesis, drug screening, and cell replacement therapies in future studies.

The ability and efficiency of mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs) to yield retinal cell types in a directed, stepwise manner was tested. hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes. Such methods represent a promising new approach for retinal stem cell research, especially translational applications., The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. Significance In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs), followed by the differentiation of these cells toward a retinal lineage, including photoreceptors, retinal ganglion cells, and retinal pigment epithelium, has been demonstrated. The use of mRNA reprogramming to yield pluripotency represents a unique ability to derive pluripotent stem cells without the use of DNA vectors, ensuring the lack of genomic integration and constitutive expression. The studies reported in the present article serve to establish a more reproducible system with which to derive retinal cell types from hiPSCs through the prevention of genomic integration of delivered genes and should also eliminate the risk of constitutive expression of these genes. Such ability has important implications for the study of, and development of potential treatments for, retinal degenerative disorders and the development of novel therapeutic approaches to the treatment of these diseases.

Retinal ganglion cells (RGCs) are a heterogeneous group of cells, comprised of numerous subpopulations, that work together to send visual information to the brain. In numerous blinding disorders termed optic neuropathies, RGCs are the main cell type affected leading to degeneration of these cells and eventual loss of vision. Previous studies have identified and characterized RGC subtypes in numerous animal systems, with only a handful of studies demonstrating their differential loss in response to disease and injury. Thus, efforts of the current study utilized an optic nerve crush (ONC) model to characterize the loss of RGCs and disease phenotypes associated with this injury. Additionally, the loss of RGC subtypes including direction selective-, alpha-, and ip-RGCs following ONC was explored. Results of this study demonstrated the differential loss of RGC subtypes with a high susceptibility for loss of alpha- and direction selective-RGCs and the preferential survival of ip-RGCs following ONC and allows for the establishment of additional studies focused on mechanisms and loss of these cells in optic neuropathies. Additionally, these results put important emphasis on the development of therapeutics targeted at the loss of specific subtypes as well as cellular replacement following injury and disease.

Human pluripotent stem cells (hPSCs) provide unprecedented access to the earliest stages of retinogenesis that remain inaccessible to investigation, thereby serving as powerful tools for studies of retinal development. Additionally, the ability to derive hPSCs from patient sources allows for the modeling of retinal degenerative diseases in vitro, with the potential to facilitate cell replacement strategies in advanced stages of disease. For these purposes, many studies over the past several years have directed the differentiation of hPSCs to generate retinal cells using stochastic methods of differentiation, yielding all major cell types of the retina. In particular, these studies have favored the derivation of RPE, photoreceptors, and more recently retinal ganglion cells for disease modeling, drug screening as well as cell replacement purposes. More recently, advances in retinal differentiation methods have led to the generation of three-dimensional retinal organoids that recapitulate key developmental and morphological features of the retina, including the stratified organization of retinal cells into a tissue-like structure. This review provides an overview of retinal differentiation from hPSCs and their potential use for studies of retinogenesis as well as diseases that affect the retina.

Objective This study aimed to use a systematic approach to evaluate the current utilization, safety, and effectiveness of cell therapies for neurological diseases in human. And review the present regulations, considering United States (US) as a representative country, for cell transplantation in neurological disease and discuss the challenges facing the field of neurology in the coming decades. Methods A detailed search was performed in systematic literature reviews of cellular-based therapies in neurological diseases, using PubMed, web of science, and clinical trials. Regulations of cell therapy products used for clinical trials were searched from the Food and Drug Administration (FDA) and the National Institutes of Health (NIH). Results Seven most common types of cell therapies for neurological diseases have been reported to be relatively safe with varying degrees of neurological recovery. And a series of regulations in US for cellular therapy was summarized including preclinical evaluations, sourcing material, stem cell manufacturing and characterization, cell therapy product, and clinical trials. Conclusions Stem cell-based therapy holds great promise for a cure of such diseases and will value a growing population of patients. However, regulatory permitting activity of the US in the sphere of stem cells, technologies of regenerative medicine and substitutive cell therapy are selective, theoretical and does not fit the existing norm and rules. Compiled well-defined regulations to guide the application of stem cell products for clinical trials should be formulated.

Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for the field of regenerative medicine because of their ability to differentiate into any cell type of the body. Such ability has profound implications for translational medicine, because these cells have been implicated for use in cell replacement, disease modeling, and pharmacological screening. However, the translation of established methods for deriving retinal cell types from hiPSCs has been hindered by the use of xenogeneic products for their growth and differentiation. Thus, the ability to derive retinal cell types in the absence of xenogeneic products would represent a significant advancement. The following studies were therefore undertaken to test the ability of hiPSCs to give rise to retinal cells under nonxenogeneic conditions. hiPSCs were maintained in traditional, feeder-free, or xeno-free culture conditions, and their ability to differentiate to a retinal fate was tested. Upon differentiation under all three conditions, cells acquired advancing features of retinal development, eventually yielding cell types of the mature retina. Reverse transcription-polymerase chain reaction and immunocytochemistry confirmed early trends in gene and protein expression patterns in xeno-free derived hiPSCs similar to those in cells derived in mouse embryonic fibroblasts and in feeder-free conditions. Results from this study demonstrate that hiPSCs can be maintained and directed to differentiate into retinal cell types under nonxenogeneic conditions, similar to cells derived using current xenogeneic methodologies. The demonstration of this capability will facilitate future efforts to develop hiPSC-based therapies for retinal disorders and also help to advance in vitro studies of human retinal development.

The ultimate goals for stem cell researchers are uncovering the fundamental mechanisms of cellular biology as well as developing treatments for disease and/or injury. The central nervous system (CNS) presents an unparalleled challenge for these studies due to the diversity of cell types and their interrelated functions in health and disease. Furthermore, the endpoints for determining phenotypic maturation are complicated by the fact that cells, especially neurons, express a plethora of receptors and ion channels that control excitability and plasticity which vary greatly between CNS regions. For this reason, extensive effort has been placed on developing methods to differentiate appropriate populations of cells that reside within the CNS (e.g., neuronal subtypes, astrocytes, and oligodendrocytes). These studies typically employ phenotypic validation by the use of expression analysis for various protein markers of regionalized populations, with a burgeoning effort to verify functional outcomes. Much of this work has been informed by basic developmental studies that have identified cell-intrinsic and cell-extrinsic signaling mechanisms, as well as transcriptional programs involved with cell fate specification.

Human pluripotent stem cells have the potential to provide comprehensive model systems for the earliest stages of human ontogenesis. To serve in this capacity, these cells must undergo a targeted, stepwise differentiation process that follows a normal developmental timeline. Here we demonstrate the ability of both human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells to meet these requirements for human retinogenesis. Upon differentiation, hESCs initially yielded a highly enriched population of early eye field cells. Thereafter, a subset of cells acquired features of advancing retinal differentiation in a sequence and time course that mimicked in vivo human retinal development. Application of this culture method to a human iPS cell line also generated retina-specific cell types at comparable times in vitro. Lastly, altering endogenous signaling during differentiation affected lineage-specific gene expression in a manner consistent with established mechanisms of early neural and retinal cell fate determination. These findings should aid in the investigation of the molecular events governing retinal specification from human pluripotent stem cells.

Retinal ganglion cells (RGCs) are highly specialized neuronal cells located in the innermost layer of the retina and serve to relay visual information to the brain, with their axons collectively forming the optic nerve. Loss or damage to the RGCs results in visual impairment and ultimately blindness. Several diseases affect the RGCs exclusively, the most common being glaucoma. Even though the mechanisms of glaucoma are not fully understood, some common treatments can delay cell death. Pharmacological intervention or laser therapy is thought to reduce the intraocular pressure and therefore reduce cell damage, but ultimately these therapies are often transient and stop working. Additional strategies to rescue RGCs and prevent their loss are being explored. Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, serve as an effective in vitro model of retinal development and repair. In addition, RGCs differentiated from hPSCs can also provide an unlimited source of transplantable cells for use in cell replacement strategies. This review addresses some of the technical and clinical issues and concerns with generating bone fide RGCs in vitro. We also outline the potential applications of hPSC-derived RGCs as a useful tool in disease modeling and drug screening in order to advance knowledge of optic neuropathies.

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies.

The procedure to efficiently and reproducibly differentiate retinal cells from human pluripotent stem cells (hPSCs) is described below. Cells are taken through a stepwise protocol to direct them toward a neural fate by treatment with neural induction medium (NIM), then to a retinal fate by exposure to retinal differentiation medium (RDM). Undifferentiated hPSCs are enzymatically lifted from matrigel-coated plates and exposed to NIM in suspension. Differentiation in suspension allows the cells to form 3 dimensional aggregates. At 7 days of differentiation, aggregates are plated and attach to 6 well plates, where a neuroepithelial fate begins to be established. Upon 16 days of differentiation, neurospheres are lifted and maintained in RDM to create a three-dimensional optic vesicle-like structure. This procedure allows for the efficient and timely generation of a variety of retinal cell types, including ganglion cells, retinal pigment epithelium, as well as cone and rod photoreceptors. The use of this protocol to generate a myriad of retinal cell types facilitates in vitro studies of human retinogenesis, and will enable retinal dysfunction to be more easily studied in vitro, as well as providing a large population of cells with which to aid in drug development and patient specific therapies.

Embryonic stem (ES) cells can differentiate into many specialized cell types, including those of the nervous system. We evaluated the differentiation of enhanced green fluorescent protein (EGFP)-expressing B5 mouse ES cells in vitro and in vivo after transplantation into the eyes of mice with hereditary retinal degeneration. After neural induction with retinoic acid, the majority of cells in embryoid bodies expressed markers for neural progenitors as well as for immature and mature neurons and glial cells. When induced ES cells were plated in vitro, further differentiation was observed and the majority of cells expressed beta-III Tubulin, a marker for immature neurons. In addition, many plated cells expressed markers for mature neurons or glial cells. Four days after intravitreal transplantation into the eyes of rd1 mice (a model of rapid retinal degeneration), donor cells appeared attached to the vitreal surface of the retina. After 6 weeks in vivo, most transplanted cells remained adherent to the inner retinal surface, and some donor cells had integrated into the retina. Transplanted cells exhibited some properties typical of neurons, including extensive process outgrowth with numerous varicosities and expression of neuronal and synaptic markers. Therefore, after induction B5 ES cells can acquire the morphologies of neural cells and display markers for neuronal and glial cells in vitro and in vivo. Furthermore, when placed in the proper microenvironment ES-derived neural precursors can associate closely with or migrate into nervous tissue where differentiation appears to be determined by cues provided by the local environment, in this case the degenerating neural retina.

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies.

The successful culture and characterization of human embryonic stem cells (hESCs) in 1998 [1] made unlimited human cell manufacture a plausible goal, ushering in a new era of science and technology, and promising to change the face of medicine. More than a decade of research later, significant advancements have been made in our understanding of the factors that govern hESC pluripotency and lineage-specific differentiation. Additional investigations into the safety and efficacy of transplanted hESC progeny in animal models of neural injury and retinal degenerative disease have brought the field to the brink of clinical trials. However, no sooner did the scientific, medical and lay communities become familiar with hESCs than a new source of pluripotent stem cells (PSCs) appeared on the scene. In 2007, the Yamanaka and Thomson laboratories independently published methods whereby human somatic cells could be reprogrammed to a pluripotent state by misexpressing a surprisingly small number of genes [2,3]. The resulting human induced PSCs (hiPSCs) appeared to have all the desirable attributes of hESCs without the ethical issues surrounding the use of human embryos. Furthermore, hiPSCs could be derived from individual patients, making it possible to develop customized stem cell therapies and generate disease-specific stem cell lines. Now that a number of laboratories have had the opportunity to evaluate both sources of PSCs, it is reasonable to examine the early findings, and ask how hiPSCs and hESCs have compared with regard to their capacity for directed cell differentiation. While several cell types could be used as readouts for such a comparison, the retinal lineage possesses a number of features that qualifies it as a robust and reliable barometer to assess the efficiency of targeted hESC and hiPSC differentiation. The retina has long served as a model of neural development owing to its accessibility, relatively simple architecture and conserved developmental program, as well as the availability of specific markers for some of its cellular constituents, such as photoreceptors and retinal pigment epithelium (RPE). Owing in large part to these advantages, a tremendous amount of information is available regarding the mechanisms, sequence of events and time course of vertebrate retinogenesis. Such knowledge is useful when evaluating the ability of PSC lines to differentiate along the retinal lineage, and, more importantly, for intervening when the process veers from the desired course. Numerous groups, including our own, have demonstrated that differentiating hESCs mimic the stepwise development of retinal cells in vivo [4–6]. Furthermore, hESCs appear to respond to secreted morphogens in a manner predicted by studies of vertebrate neural induction and retinogenesis. In particular, blockade of bone morphogenetic protein and canonical Wnt signaling is known to be important for neural and retinal patterning, and many retinal differentiation protocols call for antagonists of one or both of these pathways to be included in the culture medium [4,6]. Differentiation protocols that do not add bone morphogenetic protein or Wnt inhibitors likely rely upon their endogenous expression and secretion in culture, which could, at least partially, account for the observed ‘default’ production of early anterior neural and retinal cells [5]. In fact, numerous hESC lines, including the widely used H1 (WA01) and H9 (WA09) WiCell lines, generate anterior neuroectodermal derivatives with little, if any, manipulation of culture conditions. For example, overgrown hESC cultures will undergo spontaneous differentiation and generate patches of pigmented RPE, often in large abundance [7].

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs), but little is known about SSEA-5 expression in other primitive tissues (e.g., human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors, cell lines, teratomas, and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors, such as embryonal carcinoma. In tumor cell lines, SSEA-5 was highly immunoreactive in Capan-1 cells, while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs, with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use.

Regeneration of the nervous system requires either the repair or replacement of nerve cells that have been damaged by injury or disease. While lower organisms possess extensive capacity for neural regeneration, evolutionarily higher organisms including humans are limited in their ability to regenerate nerve cells, posing significant issues for the treatment of injury and disease of the nervous system. This chapter focuses on current approaches for neural regeneration, with a discussion of traditional methods to enhance neural regeneration as well as emerging concepts within the field such as stem cells and cellular reprogramming. Stem cells are defined by their ability to self-renew as well as their ability to differentiate into multiple cell types, and hence can serve as a source for cell replacement of damaged neurons. Traditionally, adult stem cells isolated from the hippocampus and subventricular zone have served as a source of neural stem cells for replacement purposes. With the advancement of pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs), new and exciting approaches for neural cell replacement are being developed. Furthermore, with increased understanding of the human genome and epigenetics, scientists have been successful in the direct genetic reprogramming of somatic cells to a neuronal fate, bypassing the intermediary pluripotent stage. Such breakthroughs have accelerated the timing of production of mature neuronal cell types from a patient-specific somatic cell source such as skin fibroblasts or mononuclear blood cells. While extensive hurdles remain to the translational application of such stem cell and reprogramming strategies, these approaches have revolutionized the field of regenerative biology and have provided innovative approaches for the potential regeneration of the nervous system.

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine.

A central issue in stem cell biology is the determination of function and activity of differentiated stem cells, features that define the true phenotype of mature cell types. Commonly, physiological mechanisms are used to determine the functionality of mature cell types, including those of the nervous system. Calcium imaging provides an indirect method of determining the physiological activities of a mature cell. Camgaroos are variants of yellow fluorescent protein that act as intracellular calcium sensors in transfected cells. We expressed one version of the camgaroos, Camgaroo-2, in mouse embryonic stem (ES) cells under the control of the CAG promoter system. Under the control of this promoter, Camgaroo-2 fluorescence was ubiquitously expressed in all cell types derived from the ES cells that were tested. In response to pharmacological stimulation, the fluorescence levels in transfected cells correlated with cellular depolarization and hyperpolarization. These changes were observed in both undifferentiated ES cells as well as ES cells that had been neurally induced, including putative neurons that were differentiated from transfected ES cells. The results presented here indicate that Camgaroo-2 may be used like traditional fluorescent proteins to track cells as well as to study the functionality of stem cells and their progeny.

During development of the central nervous system, stem and progenitor cell proliferation and differentiation are controlled by complex inter- and intracellular interactions that orchestrate the precise spatiotemporal production of particular cell types. Within the embryonic retina, progenitor cells are located adjacent to the retinal pigment epithelium (RPE), which differentiates prior to the neurosensory retina and has the capacity to secrete a multitude of growth factors. We found that secreted proteinaceous factors in human prenatal RPE conditioned medium (RPE CM) prolonged and enhanced the growth of human prenatal retinal neurospheres. The growth-promoting activity of RPE CM was mitogen-dependent and associated with an acute increase in transcription factor phosphorylation. Expanded populations of RPE CM-treated retinal neurospheres expressed numerous neurodevelopmental and eye specification genes and markers characteristic of neural and retinal progenitor cells, but gradually lost the potential to generate neurons upon differentiation. Misexpression of Mash1 restored the neurogenic potential of long-term cultures, yielding neurons with phenotypic characteristics of multiple inner retinal cell types. Thus, a novel combination of extrinsic and intrinsic factors was required to promote both progenitor cell proliferation and neuronal multipotency in human retinal neurosphere cultures. These results support a pro-proliferative and antiapoptotic role for RPE in human retinal development, reveal potential limitations of human retinal progenitor culture systems, and suggest a means for overcoming cell fate restriction in vitro. Disclosure of potential conflicts of interest is found at the end of this article.

Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type–specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons.

Many systemic and eye-specific genetic disorders are accompanied by retinal degenerations that lead to blindness. In some of these diseases retinal degeneration occurs early in life and is quite rapid, whereas in other disorders, retinal degeneration starts later and progresses very slowly. At present, no therapies are available to patients for preventing or reversing the retinal degeneration that occurs in these diseases. Implantation of neural progenitor cells into the eye may be a means by which to retard or even reverse degeneration of the retina. To evaluate the potential of neural precursor cell implantation for treating retinal degenerative disorders, neuralized mouse embryonic stem cells from green fluorescent protein (GFP) transgenic mice were administered intravitreally to normal mice, mice with early retinal degeneration, and mice with slowly progressing retinal degeneration. In normal mice, the donor cells remained in the vitreous cavity and did not associate with the host retina. In mice with early retinal degeneration, implantation of the neural precursors was performed after the degeneration was almost complete. In these animals, the donor cells primarily associated closely with the inner surface of the retina, although a small fraction of donor cells did integrate into the host retina. Donor cells implanted in mice with slowly progressing retinal degeneration also associated with the inner retinal surface, but many more of the cells integrated into the retina. These findings indicate the importance of host tissue-donor cell interactions in determining the fate of implanted neural precursor cells. These interactions will be a major consideration when devising strategies for using cell implantation therapies for neurodegenerative disorders.

Crustacean muscles are unique in their innervation by both excitatory and inhibitory neurons; therefore, they exhibit polyneuronal and multiterminal innervation. Because excitatory motoneurons are broadly divided into phasic and tonic types, we hypothesized that inhibitory neurons would follow a similar dichotomy. The abdominal extensor muscles in crayfish are separated into parallel deep and superficial bundles; the former has fast muscle fibers innervated by phasic excitatory motoneurons, and the latter has slow fibers supplied by tonic excitatory motoneurons. Each muscle also is innervated by a single, separate inhibitory neuron that uses γ-aminobutyric acid (GABA) as the inhibitory neurotransmitter. The pattern of axonal branching by the separate inhibitory axons in phasic and tonic abdominal extensor muscles was visualized with confocal microscopy in preparations labeled for GABA-like immunoreactivity. Initial observations indicated that the phasic muscle was covered by extensive GABAergic, filiform axon terminals, whereas innervation of the tonic muscle was comprised of more localized and varicose terminals. With quantitative analyses, we found that the phasic axon has a more highly branched nature than the tonic in first- and second-order branches. The phasic axon branches also were significantly longer than the tonic branches in the second- and third-order branches. Synaptic varicosities in the phasic branches were smaller and less frequent than those in the tonic branches. The fine structure of the inhibitory nerve terminals near synaptic contacts examined with thin-serial-section electron microscopy revealed distinct differences between the phasic system and the tonic system. The phasic terminals were smaller in cross-sectional area than the tonic terminals, and they had smaller synapses and fewer mitochondria. The presynaptic active zone dense bodies were similar in length and number between phasic and tonic synapses. However, their number per synaptic area was two-fold higher in phasic synapses compared with tonic synapses because of the smaller size of the phasic synapses. Thus, within the same neuromuscular system, inhibitory synaptic terminals revealed unique phasic and tonic identities similar to those observed for the excitatory axons. J. Comp. Neurol. 435:283–290, 2001. © 2001 Wiley-Liss, Inc.

The use of time dependent covariates has allowed for incorporation into analysis of survival data intervening events that are binary and non-reversible (for example, heart transplant, initial response to chemotherapy). We can represent this type of intervening event as a three-state stochastic process with a starting state (S), an intervening state (I), and an absorbing state (D), which usually represents death. In this paper we present three procedures for calculating survivorship functions which attempt to display the prognostic significance of the time dependent covariate. The first method compares survival from baseline for the two possible paths through the stochastic process; the second method compares overall survival to survival with state I removed from the process; and, the third method compares survival for those already in state I at a landmark time x to those in state S at time x who will never enter state I. We develop discrete hazard estimates for the survival curves associated with the three methods. Two examples illustrate how these methods can yield different results and in which situations one might employ each of the three methods. Extensions to applications with reversible binary time dependent covariates and models with both baseline and time dependent covariates are suggested.