An Advocacy for the Use of 3D Stem Cell Culture Systems for the Development of Regenerative Medicine: An Emphasis on Photoreceptor Generation

The availability of stem cells is of great promise to study early developmental stages and to generate adequate cells for cell transfer therapies. Although many researchers using stem cells were successful in dissecting intrinsic and extrinsic mechanisms and in generating specific cell phenotypes, few of the stem cells or the differentiated cells show the capacity to repair a tissue. Advances in cell and stem cell cultivation during the last years made tremendous progress in the generation of bona fide differentiated cells able to integrate into a tissue after transplantation, opening new perspectives for developmental biology studies and for regenerative medicine. In this review, we focus on the main works attempting to create in vitro conditions mimicking the natural environment of CNS structures such as the neural tube and its development in different brain region areas including the optic cup. The use of protocols growing cells in 3D organoids is a key strategy to produce cells resembling endogenous ones. An emphasis on the generation of retina tissue and photoreceptor cells is provided to highlight the promising developments in this field. Other examples are presented and discussed, such as the formation of cortical tissue, the epithelial gut or the kidney organoids. The generation of differentiated tissues and well-defined cell phenotypes from embryonic stem (ES) cells or induced pluripotent cells (iPSCs) opens several new strategies in the field of biology and regenerative medicine. A 3D organ/tissue development in vitro derived from human cells brings a unique tool to study human cell biology and pathophysiology of an organ or a specific cell population. The perspective of tissue repair is discussed as well as the necessity of cell banking to accelerate the progress of this promising field

Timing the Generation of Distinct Retinal Cells by Homeobox Proteins

The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of “sensors” in which green fluorescent protein translation is under control of the 3′ untranslated region (UTR), we found that the 3′ UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities

Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration

The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation

Ruolo del ciclo cellulare nel differenziamento retinico dei vertebrati

La generazione di tipi di neuroni diversi secondo una specifica sequenza temporale è fondamentale per la formazione di strutture neurali stratificate quali corteccia cerebrale e retina. La struttura finale della retina di vertebrato è costituita da sei principali tipi di cellule nervose (gangliari, amacrine, bipolari, orizzontali, bastoncelli e coni) e un tipo di glia (glia di Müller) distribuiti in tre strati cellulari distinti. I diversi tipi cellulari sono generati (cioè i loro progenitori cellulari escono dal ciclo cellulare) in modo temporalmente ordinato: prima le cellule gangliari, seguite da coni, amacrine ed orizzontali, bastoncelli, bipolari ed infine glia di Müller. Tuttavia non è ancora chiaro se la progressione del ciclo cellulare sia veramente necessaria ai cambiamenti di competenza differenziativa dei progenitori cellulari retinici (RPCs). Nella mia tesi ho utilizzato il gene antiproliferativo Xgadd45γ come strumento per analizzare gli effetti del blocco del ciclo cellulare sulla retinogenesi. Mediante ibridazione in situ ho evidenziato che Xgadd45γ è espresso nei territori in cui i RPCs escono dal ciclo cellulare ed iniziano ad differenziarsi. Ho poi condotto numerosi esperimenti funzionali mediante sovraespressione di Xgadd45γ in cloni cellulari isolati di RPCs, iniettando vettori di espressione a DNA in embrione a stadio di 4 cellule, o lipofettando in vivo vescicole ottiche. La sovraespressione di Xgadd45γ inibisce il differenziamento di tipi cellulari retinici tardivi come bastoncelli e cellule bipolari. Questo effetto ¨¨ opposto a quello generato dalla progressione forzata del ciclo cellulare mediante lipofezione di cdk2/ciclinaA2. Una domanda che mi sono posta è se la progressione del ciclo cellulare sia necessaria per l'espressione dei geni del differenziamento dei vari tipi cellulari. Il blocco della progressione del ciclo, effettuato sia mediante sovraespressione di Xgadd45γ sia con idrossiurea/afidicolina, inibisce l'espressione di geni di specifiche competenze differenziative quali Xbh1 (cellule gangliari), Xotx5 (fotorecettori) Xotx2 e Xchx10 (cellule bipolari), ma non del gene proneurale Xath5 o del marcatore retinico Xrx1. Inoltre RPCs forzati a dividersi mediante lipofezione di cdk2/ciclinaA2 generano poche cellule bipolari (tardive) quando Xotx2 è funzionalmente inattivato. I miei risultati suggeriscono che la progressione del ciclo cellulare dei RPCs sia necessaria per l'espressione di geni chiave che regolano la competenza differenziativa, e di conseguenza per la generazione dei tipi cellulari tardivi della retina matura

The generation of cell diversity in the vertebrate neural retina

The nervous system is an extraordinary array of different cell types that are generated at predictable times in developing brain structures. Several studies on how this variety is achieved, have revealed that for any specific cell type a combination of both intrinsic and extrinsic signals is required. The vertebrate retina is a very useful model to investigate the generation of such cell diversity. We demonstrated that retinal progenitors exit from the cell cycle in distinct waves, thus giving rise to the cell types they have competence to generate in that specific retinogenetic time. This means that, each retinal cell type has a retinogenetic timing to withdraw from the cell cycle. In the vertebrate retina, homeobox genes play crucial role in establishing different cell identities. Basing our studies on this knowledge, in this thesis work we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. We found that the three Xenopus homeobox genes Xotx5b, Xvsx1 and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1, Xotx2). Furthermore, by in vivo lipofection of “sensors” in which GFP translation is under control of the 3’ untranslated region (UTR), we found that the 3’UTRs of Xotx5b, Xvsx1 and Xotx2 are sufficient to drive a spatio-temporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequence, respectively. This is the first evidence that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities. Finally our observations suggest that this clock measures cell cycle length and that translational inhibitors are part of the clock machinery, while its molecular nature is completely unknown. We then focused our attention to find out how cell cycle progression can remove the translational inhibition over developmental time. Using an in silico approach, we found that the 3’UTR of Xotx5b, Xvsx1, and Xotx2 contain widley dispersed candidate microRNAs (miRNAs) domains for 42 distinct miRNAs. To assay the role of miRNAs during retinal development, we performed a high throughput analysis by microarrays followed by whole mount in situ hybridization. We found that several miRNAs are expressed in the frog developing retina and that their expression pattern changes during retinogenesis. Subsequently, we performed loss of function experiments on selected miRNAs (222, 155, 129). The inhibition of each miRNA caused an increase in the proportion of bipolar cells. This could be due to an increase in the translation of the corresponding target mRNAs. To investigate the role of mature miRNAs in regulating cell proliferation, survival and differentiation of retinal cells, we downregulated dicer activity in Xenopus embryos by antisense morpholino (Mo) oligonucleotides. The RNaseIII enzyme Dicer has been implicated in the maturation of most miRNAs. This means that blocking Dicer function removes mature miRNAs from a cell. The eyes of Mo-injected embryos (morphants) at the stage of swimming tadpole (st. 42) showed several defects such as: a reduced eye size, an increased retinal cell death, an incomplete retinal lamination and an increase of retinal cycling cells. Whereas Xotx5b and Xotx2 mRNAs were expressed from early developmental stages in morphants, the onset of both Xotx5b and Xotx2 protein detection was delayed in morphants compared to controls. Thus, dicer inactivation affects the timing of retinal differentiation by delaying progenitor cell divisions and the translation of key genes required for the generation of late retinal cell types. Our data suggest a role for miRNAs in the regulation of the homeobox genes translation, but do not rule out the possibility that specific RNA-binding proteins (RBPs) might also be involved. Our idea is that miRNAs could co-operate with RBPs, using a combinatorial and co-operative code, to regulate gene expression

Derivation of Traceable and Transplantable Photoreceptors from Mouse Embryonic Stem Cells

Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas

The <i>Xenopus Xvsx1</i> Homeobox Gene Supports Bipolar Cell Fate

<div><p>(A–C) Sections of st. 42-lipofected retinas. GFP (green) traces lipofection. (A) Example of control lipofection. (B and C) Example of <i>Xvsx1</i> lipofection. (C) Immunostaining (red fluorescence) with amacrine antibodies panel (anti-5-HT, anti-GABA, anti-tyrosine hydroxilase), labeling the main classes of amacrine cells at this developmental time [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040272#pbio-0040272-b047" target="_blank">47</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040272#pbio-0040272-b048" target="_blank">48</a>] ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. <i>Xvsx1</i> lipofection (B) increases the proportion of INL cells and decreases the proportion of ONL cells compared to control (A). The majority of the <i>Xvsx1</i>-lipofected cells in the INL are not stained either by amacrine markers (C), or by the horizontal marker <i>prox1</i> (not shown).</p> <p>(D) Statistical analysis showing the proportion of lipofected cell types. Cell types were identified as described [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040272#pbio-0040272-b014" target="_blank">14</a>]. Error bars indicate standard error of the mean. <i>Xvsx1</i> misexpression increases the proportion of bipolar cells (from 33% of control to 55%, student's <i>t</i>-test, <i>p</i> = 0.000043), mainly at the expense of photoreceptors (from 29% to 14%, student's <i>t</i>-test, <i>p</i> = 0.000011).</p></div

The 3′ UTRs of <i>Xotx5b, Xvsx1,</i> and <i>Xotx2</i> mRNAs Direct Time-Dependent Inhibition of Translation

<div><p>(A) Examples of time-lapse imaging of lipofected retinas. Times are calculated starting from st. 30 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040272#pbio-0040272-b022" target="_blank">22</a>] (which corresponds to time 0). To better visualize GFP, pigmentation was abolished as described [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040272#pbio-0040272-b043" target="_blank">43</a>]. Each micro-photograph shows the entire area of a lipofected eye and is focused on lipofected cells of the neural retina. Red arrows point to lipofected clones of cells.</p> <p>(B) Statistical analysis of 68 records. Bars express the proportion of lipofected retinas in which GFP was first detectable at a given time. Number of retinas examined is indicated by <i>n.</i></p></div

The 3′ UTRs of <i>Xotx5b, Xvsx1,</i> and <i>Xotx2</i> mRNA Direct Cell Type–Specific Inhibition of Translation

<div><p>(A) Detection of sensor mRNAs (Fast Red), sensor GFP protein (green immunodetection), or co-detection of both mRNA and protein (merge, yellow), in cells of mature retinas (st. 42) lipofected with GFP control vector, or UTR-carrying sensor vectors (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040272#s3" target="_blank">Materials and Methods</a>). Unlike control GFP, sensor GFP translation is detectable (arrowheads) mainly in photoreceptors (<i>Xotx5b</i> sensor) or bipolar cells (<i>Xvsx1</i> and <i>Xotx2</i> sensors). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.</p> <p>(B) Bars show the proportion of sensor-translating/sensor-transcribing cell types. Number of cells is indicated by <i>n.</i> Error bars represent standard error of the mean. Single asterisk indicates <i>p</i> ≤ 0.05; double asterisk indicates <i>p</i> ≤ 0.01 (student's <i>t</i>-test).</p></div