Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

Bertacchi, Michele; Lupo, Giuseppe; Pandolfini, Luca; Casarosa, Simona; D’Onofrio, Mara; Pedersen, Roger A.; Harris, William A.; Cremisi, Federico

doi:10.1016/j.stemcr.2015.08.011

Cited by 16 publications

(14 citation statements)

References 38 publications

(89 reference statements)

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“…Moreover, zebrafish and mice with Nodal mutations display defects in eye development such as cyclopia as well as loss of ath5 ( ATOH7 ) expression , an essential gene for RGC development . Furthermore, recently a role for Nodal/Activin signaling in establishment of the eye field has been described in hESC and mESC differentiation and addition of IDE1, another Nodal agonist molecule similar to IDE2, has been used to enhance stem cell differentiation to RPE . Surprisingly, we noticed that DSM alone decreased RGC differentiation, perhaps due to its weaker activity against Activin/Nodal .…”

Section: Discussionmentioning

confidence: 77%

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells

Sluch

Chamling

Liu

et al. 2017

Stem Cells Translational Medicine

100

151

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Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularlyinterspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for largescale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of largescale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1972-1986 SIGNIFICANCE STATEMENTPluripotent stem cells provide access to a large variety of human cell types. However, stem cell culture is often heterogeneous and efficient differentiation and purification of stem cell-derived cells are limited to relatively few examples. Via genetic engineering, we have generated stem cell reporter lines that enable the detection and purification of retinal ganglion cells (RGCs), an essential cell type for vision. Through modulation of known signaling pathways, we report an improved RGC differentiation protocol for high yields of purified RGCs. We also describe an siRNA protocol for exploration of signaling pathways in human RGCs.

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Section: Discussionmentioning

confidence: 77%

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells

Sluch

Chamling

Liu

et al. 2017

Stem Cells Translational Medicine

100

151

View full text Add to dashboard Cite

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“…Our finding that activin A causes similar effects on some, but not all, non-retinal populations suggests that activin alone is insufficient to induce these gene expression changes ( Figure S1 ). Other studies on neuralization and eye field specification in differentiating ESCs have shown that activin signaling coordinates with bFGF, BMP, and Wnt pathways ( Bertacchi et al., 2015 , Lupo et al., 2013 ).…”

Section: Discussionmentioning

confidence: 99%

“…Activin A treatment of differentiating ESCs caused increased expression of eye field and RPC markers ( Bertacchi et al., 2015 ). We investigated the effect of activin signaling on ESC-derived cultures that had reached the RPC stage, a time frame that corresponds to in vivo dates at which expression of activin A and its receptors were detected in the embryonic eye and retina.…”

Section: Discussionmentioning

confidence: 99%

“…Because the effects of activin A are dependent on the time point at which it is introduced, we propose a schematic of development in which activin signaling first maintains pluripotency and therefore needs to be inhibited for differentiation to begin ( Wong et al., 2015 , Zhou et al., 2015 ) ( Figure 7 C1). Following this, it promotes eye field formation and generation of RPCs ( Bertacchi et al., 2015 ) ( Figure 7 C2). After the RPC pool has been established, our work shows that activin supports the transition from RPCs into photoreceptor precursors ( Figure 7 C3 and C4).…”

Section: Discussionmentioning

confidence: 99%

“…Activin promotes eye field formation in ESCs ( Bertacchi et al., 2015 , Lupo et al., 2013 ) and generation of mature photoreceptors in primary rodent retina cultures ( Davis et al., 2000 ), but also prevents differentiation of pluripotent stem cells by regulating the expression of key stem cell genes such as Oct4 and Nanog ( Beattie et al., 2005 , Sun et al., 2014 , Vallier et al., 2009 , Xu et al., 2008 ) and needs to be inhibited in ESCs and blastula stage embryos to allow initial differentiation ( Wong et al., 2015 , Zhou et al., 2015 ). Thus, while activin is involved in the earliest and last steps of retinal development, there is currently no information about its role in intermediate steps.…”

Section: Introductionmentioning

confidence: 99%

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Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation

Popova

Barnstable

2017

Stem Cell Reports

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SummaryIn vitro differentiation of mouse embryonic stem cells (ESCs) into retinal fates can be used to study the roles of exogenous factors acting through multiple signaling pathways during retina development. Application of activin A during a specific time frame that corresponds to early embryonic retinogenesis caused increased generation of CRX+ photoreceptor precursors and decreased PAX6+ retinal progenitor cells (RPCs). Following activin A treatment, SMAD2/3 was activated in RPCs and bound to promoter regions of key RPC and photoreceptor genes. The effect of activin on CRX expression was repressed by pharmacological inhibition of SMAD2/3 phosphorylation. Activin signaling through SMAD2/3 in RPCs regulates expression of transcription factors involved in cell type determination and promotes photoreceptor lineage specification. Our findings can contribute to the production of photoreceptors for cell replacement therapy.

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Generation of Photoreceptor Precursors from Mouse Embryonic Stem Cells

Barnstable

2017

Stem Cell Rev and Rep

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Embryonic stem cell (ESC) differentiation can be used to model development and to produce transplantable cells of the desired phenotype. ESCs can reproducibly generate retinal cells but the derivation of photoreceptor precursors is variable and depends on an array of exogenous factors and intrinsic cell-cell interactions. In this work, we have defined the use of exogenous signaling factors, dissociation, and adherent versus 3-dimensional (3D) conditions on the derivation of retinal cells from pluripotent mouse ESCs. Differentiation protocols were chosen based on the developmental stage and cell population of interest and evaluated by expression of developmental stage- and lineage-specific marker genes. We present a relatively simple protocol that guides differentiating ESCs through stages that correspond to the sequence of in vivo developmental events and is optimized for studying the time frame between eye field formation and photoreceptor precursor development in the equivalent of embryonic retina. Step-wise exposure of adherent cultures to exogenous factors facilitated expression of eye field transcription factors and limited non-specific differentiation. Dissociation after the establishment of eye field and retinal progenitor cell gene expression did not cause substantial loss in expression of markers of mature photoreceptors. Finally, 3D organoids improved expression of photoreceptor genes and region-specific architecture but required more technical manipulation. We demonstrate the usefulness of this ESC-retinal induction protocol in screening for factors that improve photoreceptor precursor yield by evaluating response to alterations in Activin signaling.

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Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

Cited by 16 publications

References 38 publications

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells

Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation

Generation of Photoreceptor Precursors from Mouse Embryonic Stem Cells

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