Chemical induction of neurogenic properties in mammalian Müller glia.

Müller glia (MG), cells that maintain homeostasis in the retina, are dormant stem cells that can regenerate neurons upon injury. However, the regenerative property of MG, which is reproducibly displayed in the lower vertebrates, is not readily observed in the mammals even upon forced expression of regulatory genes or exposure to growth factors. Here, we demonstrate a reproducible unmasking of the neurogenic properties of enriched rodent MG by serial exposure to different combinations of small molecules. The enriched MG, in response to changing culture conditions, silenced glia-specific genes and acquired transcriptional signature of neurons, accompanied by upregulation of genes known to regulate neuronal potential of MG. The MG-derived neurons expressed immunoreactivities corresponding to neuronal proteins and displayed electrophysiological features of immature neurons. Our study presents a proof of principle of pharmacological activation of neurogenic properties of mammalian MG, which may be utilized for therapeutic regeneration.

Mapping developmental trajectories and subtype diversity of normal and glaucomatous human retinal ganglion cells by single-cell transcriptome analysis.

Glaucoma is characterized by a progressive degeneration of retinal ganglion cells (RGCs), leading to irreversible vision loss. Currently, there is no effective treatment for RGC degeneration. We used a disease-in-a-dish stem cell model to examine the developmental susceptibility of RGCs to glaucomatous degeneration, which may inform on the formulation of therapeutic approaches. Here, we used single-cell transcriptome analysis of SIX6 risk allele (SIX6risk allele ) primary open angle glaucoma patient-specific and control hRGCs to compare developmental trajectories in terms of lineage- and stage-specific transcriptional signature to identify dysregulated stages/genes, and subtype composition to estimate the relative vulnerability of RGCs to degeneration because their ability to regenerate axons are subtype-specific. The developmental trajectories, beginning from neural stem cells to RGCs, were similar between SIX6risk allele and control RGCs. However, the differentiation of SIX6risk allele RGCs was relatively stalled at the retinal progenitor cell stage, compromising the acquisition of mature phenotype and subtype composition, compared with controls, which was likely due to dysregulated mTOR and Notch signaling pathways. Furthermore, SIX6risk allele RGCs, as compared with controls, expressed fewer genes corresponding to RGC subtypes that are preferentially resistant to degeneration. The immature phenotype of SIX6risk allele RGCs with underrepresented degeneration-resistant subtypes may make them vulnerable to glaucomatous degeneration.

Recapitulating developmental mechanisms for retinal regeneration.

Degeneration of specific retinal neurons in diseases like glaucoma, age-related macular degeneration, and retinitis pigmentosa is the leading cause of irreversible blindness. Currently, there is no therapy to modify the disease-associated degenerative changes. With the advancement in our knowledge about the mechanisms that regulate the development of the vertebrate retina, the approach to treat blinding diseases through regenerative medicine appears a near possibility. Recapitulation of developmental mechanisms is critical for reproducibly generating cells in either 2D or 3D culture of pluripotent stem cells for retinal repair and disease modeling. It is the key for unlocking the neurogenic potential of Müller glia in the adult retina for therapeutic regeneration. Here, we examine the current status and potential of the regenerative medicine approach for the retina in the backdrop of developmental mechanisms.

Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway.

The poor axon regeneration in the central nervous system (CNS) often leads to permanent functional deficit following disease or injury. For example, degeneration of retinal ganglion cell (RGC) axons in glaucoma leads to irreversible loss of vision. Here, we have tested the hypothesis that the mTOR pathway regulates the development of human RGCs and that its recruitment after injury facilitates axon regeneration. We observed that the mTOR pathway is active during RGC differentiation, and using the induced pluripotent stem cell model of neurogenesis show that it facilitates the differentiation, function and neuritogenesis of human RGCs. Using a microfluidic model, we demonstrate that recruitment of the mTOR pathway facilitates human RGC axon regeneration after axotomy, providing evidence that the recapitulation of developmental mechanism(s) might be a viable approach for facilitating axon regeneration in the diseased or injured human CNS, thus helping to reduce and/or recover loss of function.

miR-29c regulates neurogliogenesis in the mammalian retina through REST.

In the developing central nervous system, including its simple and accessible model retina, neurogenesis is followed by gliogenesis. However, the mechanism underlying the neurogliogenic switch remains poorly understood despite the identification of several regulatory genes, associated with the lineage identity and transition. The mechanism may involve cross talks between regulatory genes, facilitated through microRNAs. Here, we posit miR-29c as one of the regulatory miRNAs that may influence neuronal versus glial differentiation. We observed that the temporal patterns of miR-29c expression corresponded with late retinal histogenesis, the stage in the developing retina when neurogliogenic decision predominantly occurs. Examination of the effects of miR-29c on neurogliogenesis by the perturbation of function approach revealed that miR-29c preferentially facilitated differentiation of late RPCs into rod photoreceptors and bipolar cells, the late-born neurons, at the expense of Müller glia, the sole glia generated by retinal progenitor cells. We further observed that miR-29c facilitated neurogenesis and inhibited gliogenesis by regulating the expression of RE-1 silencing transcription factor (REST), which encodes a transcriptional repressor of cell cycle regulators and neuronal genes. Thus, miR-29c may influence neurogliogenic decision in the developing retina by regulating the instructive out put of a molecular axis helmed by REST.

Lin28a regulates neurogliogenesis in mammalian retina through the Igf signaling.

In the developing central nervous system (CNS) the majority of neurons are born before the generation of glia. Emerging evidence implicates heterochronic gene, Lin28 in the temporal switch between two distinct lineages. However, the respective contributions of Lin28a and Lin28b in neurogliogenesis remain poorly understood. Here, we have examined the relative involvement of Lin28a and Lin28b in mammalian retina, a simple and accessible CNS model where neurogliogenic decision largely occurs postnatally. Examination of Lin28a/b involvement during late histogenesis by the perturbation of function approaches revealed that while Lin28b did not influence differentiation in general Lin28a facilitated and antagonized the generation of neurons and glia, respectively. Silencing of Lin28a expression in vitro and its conditional deletion in vivo during early histogenesis led to premature generation of glia. The instructive role of Lin28a on neuronal differentiation was revealed by its influence to suppress glial-specific genes and directly differentiate glia along the neuronal lineage. This function of Lin28a is likely mediated through the Igf signaling, as inhibition of the pathway abrogated Lin28a-mediated neurogliogenesis. Thus, our observations suggest that Lin28a is an important intrinsic factor that acts in concert with cell-extrinsic factors like Igfs, coordinating the developmental bias of the progenitors and niche, respectively, for the successive generation of neurons and glia.

Modeling Glaucoma: Retinal Ganglion Cells Generated from Induced Pluripotent Stem Cells of Patients with SIX6 Risk Allele Show Developmental Abnormalities.

Glaucoma represents a group of multifactorial diseases with a unifying pathology of progressive retinal ganglion cell (RGC) degeneration, causing irreversible vision loss. To test the hypothesis that RGCs are intrinsically vulnerable in glaucoma, we have developed an in vitro model using the SIX6 risk allele carrying glaucoma patient-specific induced pluripotent stem cells (iPSCs) for generating functional RGCs. Here, we demonstrate that the efficiency of RGC generation by SIX6 risk allele iPSCs is significantly lower than iPSCs-derived from healthy, age- and sex-matched controls. The decrease in the number of RGC generation is accompanied by repressed developmental expression of RGC regulatory genes. The SIX6 risk allele RGCs display short and simple neurites, reduced expression of guidance molecules, and immature electrophysiological signature. In addition, these cells have higher expression of glaucoma-associated genes, CDKN2A and CDKN2B, suggesting an early onset of the disease phenotype. Consistent with the developmental abnormalities, the SIX6 risk allele RGCs display global dysregulation of genes which map on developmentally relevant biological processes for RGC differentiation and signaling pathways such as mammalian target of rapamycin that integrate diverse functions for differentiation, metabolism, and survival. The results suggest that SIX6 influences different stages of RGC differentiation and their survival; therefore, alteration in SIX6 function due to the risk allele may lead to cellular and molecular abnormalities. These abnormalities, if carried into adulthood, may make RGCs vulnerable in glaucoma. Stem Cells 2017;35:2239-2252.

A Co-culture Model for Determining the Target Specificity of the de novo Generated Retinal Ganglion Cells.

In glaucoma, the output neurons of the retina, the retinal ganglion cells (RGCs), progressively degenerate, leading to irreversible blindness (Ahram et al., 2015). The ex vivo stem cell method to replace degenerated RGCs remains a potentially viable approach (Levin et al., 2004). However, the success of the approach depends upon the ability of the de novo generated RGCs to connect over the long distance with specific targets in the central visual pathway. Here, we describe a protocol to examine the target specificity of the de novo generated RGCs using a co-culture approach where the RGCs neurites are allowed to choose between specific (superior colliculus; SC) and non-specific (inferior colliculus; IC) tectal targets.

Generation of Functional Human Retinal Ganglion Cells with Target Specificity from Pluripotent Stem Cells by Chemically Defined Recapitulation of Developmental Mechanism.

Glaucoma is a complex group of diseases wherein a selective degeneration of retinal ganglion cells (RGCs) lead to irreversible loss of vision. A comprehensive approach to glaucomatous RGC degeneration may include stem cells to functionally replace dead neurons through transplantation and understand RGCs vulnerability using a disease in a dish stem cell model. Both approaches require the directed generation of stable, functional, and target-specific RGCs from renewable sources of cells, that is, the embryonic stem cells and induced pluripotent stem cells. Here, we demonstrate a rapid and safe, stage-specific, chemically defined protocol that selectively generates RGCs across species, including human, by recapitulating the developmental mechanism. The de novo generated RGCs from pluripotent cells are similar to native RGCs at the molecular, biochemical, functional levels. They also express axon guidance molecules, and discriminate between specific and nonspecific targets, and are nontumorigenic. Stem Cells 2017;35:572-585.

Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders.

BACKGROUND & OBJECTIVES: Human embryonic stem cell (hESC) lines are commonly maintained on inactivated feeder cells, in the medium supplemented with basic fibroblast growth factor (bFGF). However, limited availability of feeder cells in culture, and the high cost of growth factors limit their use in scalable expansion of hESC cultures for clinical application. Here, we describe an efficient and cost-effective feeder and bFGF-free culture of hESCs using conditioned medium (CM) from immortalized feeder cells. METHODS: KIND-1 hESC cell line was cultured in CM, collected from primary mouse embryonic fibroblast, human foreskin fibroblast (HFF) and immortalized HFF (I-HFF). Pluripotency of KIND-1 hESC cell line was confirmed by expression of genes, proteins and cell surface markers. RESULTS: In culture, these cells retained normal morphology, expressed all cell surface markers, could differentiate to embryoid bodies upon culture in vitro. Furthermore, I-HFF feeder cells without supplementation of bFGF released ample amount of endogenous bFGF to maintain stemness of hESC cells. INTERPRETATION & CONCLUSIONS: The study results described the use of CM from immortalized feeder cells as a consistent source and an efficient, inexpensive feeder-free culture system for the maintenance of hESCs. Moreover, it was possible to maintain hESCs without exogenous supplementation of bFGF. Thus, the study could be extended to scalable expansion of hESC cultures for therapeutic purposes.

Enhanced Reprogramming Efficiency and Kinetics of Induced Pluripotent Stem Cells Derived from Human Duchenne Muscular Dystrophy.

UNLABELLED: The generation of disease-specific induced pluripotent stem cells (iPSCs) holds a great promise for understanding disease mechanisms and for drug screening. Recently, patient-derived iPSCs, containing identical genetic anomalies of the patient, have offered a breakthrough approach to studying Duchenne muscular dystrophy (DMD), a fatal disease caused by the mutation in the dystrophin gene. However, development of scalable and high fidelity DMD-iPSCs is hampered by low reprogramming efficiency, the addition of expensive growth factors and slow kinetics of disease-specific fibroblasts. Here, we show an efficient generation of DMD-iPSCs on bFGF secreting human foreskin fibroblast feeders (I-HFF) by employing single polycistronic lentiviral vector for delivering of transcription factors to DMD patient-specific fibroblast cells. Using this method, DMD-iPSCs generated on I-HFF feeders displayed pluripotent characteristics and disease genotype with improved reprogramming efficiency and kinetics over to mouse feeders. Moreover, we were able to maintain disease-specific iPSCs without additional supplementation of bFGF on I-HFF feeders. Our findings offer improvements in the generation of DMD-iPSCs and will facilitate in understanding of pathological mechanisms and screening of safer drugs for clinical intervention. KEY WORDS: Duchenne Muscular Dystrophy, Reprogramming, Induced pluripotent Stem Cells, Immortalized Human Feeder, Basic Fibroblast Growth Factor, Stem Cell Cassette.

Continuous non-cell autonomous reprogramming to generate retinal ganglion cells for glaucomatous neuropathy.

Glaucoma, where the retinal ganglion cells (RGCs) carrying the visual signals from the retina to the visual centers in the brain are progressively lost, is the most common cause of irreversible blindness. The management approaches, whether surgical, pharmacological, or neuroprotective do not reverse the degenerative changes. The stem cell approach to replace dead RGCs is a viable option but currently faces several barriers, such as the lack of a renewable, safe, and ethical source of RGCs that are functional and could establish contacts with bona fide targets. To address these barriers, we have derived RGCs from the easily accessible adult limbal cells, reprogrammed to pluripotency by a non-nucleic acid approach, thus circumventing the risk of insertional mutagenesis. The generation of RGCs from the induced pluripotent stem (iPS) cells, also accomplished non-cell autonomously, recapitulated the developmental mechanism, ensuring the predictability and stability of the acquired phenotype, comparable to that of native RGCs at biochemical, molecular, and functional levels. More importantly, the induced RGCs expressed axonal guidance molecules and demonstrated the potential to establish contacts with specific targets. Furthermore, when transplanted in the rat model of ocular hypertension, these cells incorporated into the host RGC layer and expressed RGC-specific markers. Transplantation of these cells in immune-deficient mice did not produce tumors. Together, our results posit retinal progenitors generated from non-nucleic acid-derived iPS cells as a safe and robust source of RGCs for replacing dead RGCs in glaucoma.

Cornelia de Lange syndrome: a case study.

Cornelia de Lange syndrome (CDLS) is a relatively common multiple congenital anomaly/mental retardation disorder with an unknown genetic and molecular pathogenesis. The essential features of this developmental malformation syndrome are retardation in growth, developmental delay, various structural limb abnormalities, and distinctive facial features. Most cases are sporadic and are thought to result from a new dominant mutation. Consequently, hypotheses regarding the pathogenetic mechanisms underlying the two distinct phenotypes, classic and mild, are purely speculative. The recent discovery of molecular techniques and identification of the NIPBL gene has allowed etiologic diagnosis of this disorder. In this article, we describe a patient with CDLS in whom conventional cytogenetics, fluorescence in situ hybridization, and NIPBL gene mutation analysis determined an etiologic diagnosis, providing precise genetic counseling and facilitated the family to make an evidence-based decision for conception and also alleviated the extreme degree of anxiety associated with the thought of having a second child in this set of circumstances.