Engineering fluorescent reporters in human pluripotent cells and strategies for live imaging human neurogenesis

  1. Alwyn Dady
  2. Lindsay Davidson
  3. Nicolas Loyer
  4. Sophie Rappich
  5. Greg Findlay
  6. Timothy Sanders
  7. Jens Januschke
  8. Kate G. Storey

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Abstract

Investigation of cell behaviour and cell biological processes underlying human development is facilitated by creation of fluorescent reporters in human pluripotent stem cells, which can be differentiated into cell types of choice. Here we report use of a piggyBac transposon-mediated stable integration strategy to engineer human pluripotent stem cell reporter lines. These express a plasma membrane localised protein tagged with the fluorescent proteins eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, or the cytoskeletal protein F-tractin tagged with mKate2. Focussing on neural development these lines were used to live image and quantify cell behaviours, including cell cycle progression and cell division orientation in spinal cord rosettes. Further, lipofection-mediated introduction of piggyBac constructs into human neural progenitors labelled single cells and small cell groups within rosettes, allowing individual cell behaviours including neuronal delamination to be monitored. Finally, using the F-tractin-mKate2 hiPSC line, novel actin dynamics were captured during proliferation in cortical neural rosettes. This study presents and validates new tools and techniques with which to interrogate human cell behaviour and cell biology using live imaging approaches.

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    Reply to the reviewers

    We thank all the reviewers for their helpful and constructive comments and for their time.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):*

    Summary: Dady et al have developed fluorescent reporters to enable live imaging of cell behaviour and morphology in human pluripotent stem cell lines (PSCs). These reporters target 3 main features, the plasma membrane, nucleus and cytoskeleton. Reporter PSCs have been generated using a piggyBac transposon-mediated stable integration strategy, using a hyperactive piggyBac transposase (HyPBase). The same constructs were also used for mosaic labelling of cells within 2D cultures using lipofectamine transfection.

    The reporters used are tagged with either eGFP or mKate2 (far red) and tag the plasma membrane (pm) via the addition of a 20 amino-acid sequence from rat GAP-43 to the N-terminus of the fluorescent protein, the nucleus via Histone 2B with a laser-mediated photo-conversion option (H2B-mEos3.2), and the cytoskeleton via F-Tractin. In total, the authors produced lines with the following:

    • pm-mKate2 (far red) • pm-eGFP (green) • H2B-mEos3.2 (green to red) • F-tractin-mKate2 (far red) • H2B-mEos3.2 and pm-mKate2 (green to red, plus far red)

    The cell lines used to generate these were the human embryonic stem cell line H9 and human induced pluripotent cell line ChiPS4. The constructs were also used to label cells in a mosaic fashion, using lipofectamine transfection of the original cell lines once they had formed neural rosettes.

    Using these cells, Dady et al then performed live imaging in vitro of human spinal cord rosettes and assessed cell behaviour. In particular they analysed mitotic cleavage planes and apical positioning of neural progenitor cells (NPCs), and assessed actin dynamics within these cells. They showed a slowing of the cell cycle length after the initial expansion phase, an increase in the rate of asymmetric division of these NPCs, and abscission of the apical membrane during these divisions. The F-tractin reporter showed enrichment at the basal nuclear membrane during these cell divisions, suggested to help prevent basal chromosome displacement during mitosis.

    Major comments: The data presented are convincing and could be strengthened by the following additions and clarifications:*

    • How long do the fluorescent reports take to be visible when transfected via lipofectamine? How efficiently are they expressed? And what concentrations were tested to enable the mosaic expression presented? * We followed the manufacturer’s instructions for Lipofectamine 3000 transfection, using the protocol recommended for set up for a 6 wells plate. We detected fluorescence the following morning ~16h. We did not assess earlier time points or optimise efficiency as we observed the mosaic pattern of expression we set out to achieve, with small groups of labelled cells and single cells as shown in Figure 3 and movies 2 and 3. This information and the detailed protocol provided below are now included in the Methods section “Labelling individual cells in human spinal cord rosettes by lipofection”.

    Manufacturer’s instructions for Lipofectamine 3000 transfection (6 well plate):

    • 1 tube containing 125 ul of Opti-MEM and 7.5 ul of Lipofectamine 3000
    • 1 tube containing 250 ul of Opti-MEM with 5 ug of DNA (total mix DNAs of 2 ug/ul) and P3000 Reagent
    • Add diluted DNA to diluted Lipofectamine 3000 (Ratio 1:1) and incubate for 10 to 15 min at Room Temperature.
    • 20 ul of DNA-Lipid complex was added to neural rosettes growing in 8 well IBIDI dishes (20 ul/well).
    • The ratio of DNA (PiggyBac plasmid) and HypBase transposase was kept at 5:1 (for a final concentration of 2ug/ul).
    • Cells in IBIDI dishes were left to develop in a sterile incubator overnight and mosaic fluorescence was observed the following morning (~16h post-lipofection).
    • Will these cell lines and constructs be made publicly available after publication?*

    The cell lines can be made available: for those reporters made in the H9 WiCell line an MTA will first have to be signed between the requesting PI and WiCell and permission for us to share the line(s) confirmed by WiCell; similarly, for reporters in ChiPS4 line an MTA will first need to be signed between the requesting PI and Cellartis/TakaraBio Europe. We will need to make a charge to cover costs. Constructs will be deposited with Addgene.

    • Were the H9 and ChiPS4 lines characterised after the reporters were added to show they still proliferate/differentiate as they did prior to the reporter integration*?

    In the Results we make clear that all lines created are polyclonal, with exception of a pm-eGFP ChiPS4 line, which is a monoclonal line (lines 145-150). We do not have direct data measuring cell proliferation but collected cell passaging data for all the reporter lines. This showed that they grow to similar densities at each passage compared to the parental line (this metadata is now provided as Supplementary data 1 and is cited in the Methods, line 348).

    As a proof of principle for this approach, we created one monoclonal line from a polyclonal line ChIPS4-pm-eGFP. The latter was made by selecting an individual clone and this was then expanded and characterised for expression of pluripotency markers (immunocytochemistry data Figure S4), and the ability to differentiate into 3 germ layers (qPCR Supplementary data 1). This information is already cited in the Methods (Lines 358-362).

    • Can the novel actin dynamics described be quantified? How many cells imaged show these novel dynamics?* Some of this quantification data was already reported in the paper (in figure 4 legend and in the Methods); we have now updated this and provide the detailed metadata in an Excel spread sheet, Supplementary data 4 (cited in the Methods, line 489)

    *Minor comments:

    1. Some images in the figures and supplemental movies are low in resolution, for example the DAPI in Fig 4B, making it hard to distinguish individual cells. Please increase this.*

    We consider the DAPI labelling in Figure 4b to be clear, however, we wonder whether the reviewer was expecting to also see this combined with the other markers. We have therefore now provided these merged additional images in a revised Figure 4.

    • Please show a merge of Phalloidin and F-Tractin in Fig4, this will help the colocalization to be fully appreciated.*

    This has now been provided in revised Figure 4B.

    • Some additional annotation on the supplemental movies would be useful to indicate to the **reader exactly what cell to follow. *

    We have added indicative arrows to the movies, and note that more detailed labelling of the series of still images from these movies are provided in the main figures (Figures 3D and 4E & F).

    *Reviewer #1 (Significance (Required)):

    Human neurogenesis is currently poorly understood compared to many model systems used, yet key differences have already been identified between the human and the mouse, prompting the need for further investigation of human neural development. A major reason that human neurogenesis has been difficult to study is a lack of tools to enable cell morphology and behaviours to be analysed in real time.

    The reporters and reporter PSC lines generated by Dady et al will allow many of these cell characteristics to be observed using live imaging. For example, the morphology of neural progenitors during and after cell divisions, how the apical and basal processes and membranes are divided, and how the actin cytoskeleton helps to regulate these processes.

    *Importantly, PSC lines can be very heterogeneous, making generating reporter lines costly and time intensive. The use of these reporters with lipofectamine transfection, for a mosaic labelling, allows the visualisation of the plasma membrane, nucleus and cytoskeleton in any human PSC/NPC line, or even in human tissue cultures, without the need to generate each specific reporter line, making it a valuable tool for many labs in the field.

    We strongly agree with this final point; this is a major reason for our study.

    Reviewer #2* (Evidence, reproducibility and clarity (Required)):**

    The manuscript describes the generation of novel lines of human pluripotent stem cells bearing fluorescent reporters, engineered through piggyBac transposon-mediated integration. The cells are differentiated into neuronal organoids, allowing to capture cellular behaviors associated to cell division. A replating protocol allows the observation of aging neurons by reducing the thickness of the tissue thereby facilitating live imaging. The authors also leverage the transposon technology to create mosaically-labelled organoids which allows visualizing aspects of neuronal delamination, notably cytoskeleton dynamics. They discover an undescribed pattern of F-actin enrichment at the basal nuclear membrane prior to nuclear envelope breakdown.

    L104-109: "Moreover, the transposon system obviates drawbacks of directly engineering endogenous proteins...". Despite the risk of endogenous protein dysfunction, directly tagging allows the full regulation of gene expression (including the promoter, the enhancers and other regulatory regions rather than a strong constitutive promoter such as CAG). In addition, the number of copies integrated and the genomic regions are variable with PB, which does not reflect the endogenous expression. This could be rephrased by nuancing the advantages and drawbacks of each approach. The PiggyBac method is easier and faster, but it results in overexpression of a tagged protein that will be expressed since the hESC state and might not reflect the expression dynamics of the endogenous protein.* We agree and have now revised this in the Introduction L109-118.

    *L124-126: "To monitor cell shape and dynamics we used a plasma membrane (pm) localized protein tagged with eGFP or mKate2 (pm-eGFP or pm-mKate2)." Could the authors provide more details and a reference on the palmitoylated rat peptide use to force membrane expression? *

    This information, including the peptide sequence, is provided in the Methods (L330-331), we have now added a reference addressing its role in membrane localisation PMID: 2918027.

    L132-133: " Finally, to observe actin cytoskeletal dynamics we selected F-tractin, for its minimal impact on cytoskeletal homeostasis".

    A recent JCB paper (https://doi.org/10.1083/jcb.202409192) suggests that "F-tractin alters actin organization and impairs cell migration when expressed at high levels". Whether the overexpression of F-tractin in hESC using a CAG promoter reflects the physiological F-actin dynamics and/or if the high levels could lead to an alteration of cell behavior should be addressed or at least discussed. The paper we cite in this sentence (Belin et al 2014) evaluates F-tractin expression against other approaches to labelling and monitoring the actin cytoskeleton and concludes that in comparison F-tractin has minimal impact.

    We do appreciate that expression above the endogenous level has the potential to alter cell behaviour and have revised the paper to more explicitly acknowledge this: in the Introduction (L109-112), and in the Discussion/conclusion (L289-293) where we now note the recent advances reported in Shatskiy et al. 2025 PMID: 39928047.

    “A further potential limitation of this approach is that over-expression driven by the CAG promoter might not reflect physiological protein dynamics and/or alter cell behaviour; for example, high levels of F-Tractin can impair cell migration and induce actin bundling, interestingly, this can now be minimised by removing the N-terminal region (Shatskiy et al 2025)”.

    L146-147: "...to generate polyclonal cell lines selected for expression of easily detectable (medium level) fluorescence for live imaging studies". What are the criteria used to define medium level? Number of copies integrated into the genome? Or levels by FACS during clone selection?

    To clarify, all the lines presented here are polyclonal, except for one clonal line, pm-eGFP in ChiPS4. The numbers of copies integrated may vary from cell to cell in polyclonal lines. In this study, we selected cells for all lines with a FACS gate and this data is presented in Figure S1 (see line 147).

    L260-263: "Efficient stable integration and moderate expression levels were achieved by optimising, i) the quantity and ratio of piggyBac plasmids and transposase and ii) subsequent FACS to exclude high expressing cells, as well as iii) transfection methods, including temporally defined lipofection in hiPSC-derived tissues." The ration 5:1 is classically used for PB Transposase delivery, however there is still high variability in the number of copies integration. Lipofection in derived tissues has been shown to be challenging. Could the authors should provide quantitative data regarding the efficiency of their approaches, notably the level of mosaicism one could expect?

    We provide quantitative data for the efficiency of transfection using nucleoporation assays (FACS data presented in Supplementary figure S1), which shows more than 80-90% efficiency for eGFP in 82.82% of cells, mKate2 in 92.74% of cells, and H2B-mEos3 22.75% of cells, while 13.79% of cells co-expressed pm-Kate and H2B-mEos3.2. No comparative data regarding the efficiency of the tissue Lipofection assay was collected: our goal was to label single/small numbers of cells in order to monitor individual cell behaviours, and this “inefficient labelling” was readily achieved following the manufacturer’s instructions (please see response to Review 1 point 1), further details are now provided in the Methods.

    L191-194: "We further wished to monitor sub-cellular behaviour within the developing neuroepithelium. To achieve this, we devised a strategy to target a mosaic of cells in established neural rosettes using lipofection. PiggyBac constructs and HyPBase transposase were transfected into D8/D9 human spinal cord neural progenitors using lipofectamine (Felgner, et al., 1987)(Fig. 3A)." The mosaicism is not an all or nothing in this method but also leads to variations in expression levels among the positive cells. The protocol for lipofection could be better detailed to allow easy reproduction by other teams, and its expected efficiency should be discussed. It would be interesting to explore the relationship between individual cells phenotype and expression levels. Please see response to Reviewer 1 point 1 above for more detailed lipofection protocol which generated mosaic expression, this is now also included in the Methods. We agree that investigating the relationship between individual cell phenotypes and expression levels would be interesting, but we think this is beyond the scope of this paper.

    Additional comments: -Did the authors perform karyotyping of the hPSCs prior to use in the differentiation protocol?

    As these are polyclonal lines, we did not undertake karyotyping. This could be done for the one monoclonal line described here (pm-eGFP ChiPS4 line): we lack funds for commercial options, but we are exploring other possibilities.

    -Were pluripotency assays performed after reporter lines generation?

    These were carried out for the clonal pm-eGFP ChiPS4 line (lines 145-150). The latter was made by selecting an individual clone and this was then expanded and characterised for expression of pluripotency markers by IF (Figure S4), and the ability to differentiate into 3 germ layers by qPCR (Supplementary data 2). This information is provided in the Methods (Lines 358-362).

    *-Did the authors measure the cell proliferation rate in H2B-overexpressing cells and controls? Since H2B plays an important role in cytokinesis, it could interfere in cell division when H2B is overexpressed (see doi: 10.3390/cells8111391). *

    We did not directly measure cell division when H2B is over-expressed. However, we assessed cell -passaging time of all the transfected cell lines. This showed that they grow to similar densities at each passage compared to the parental line (this is now provided as Supplementary data 1 and is cited in the Methods, line 348). We also found no difference between apical visiting time of progenitors in spinal cord rosettes expressing pm-eGFP or H2B-mEoS3.2, further supporting the conclusion that levels of H2B-mEoS3.2 expression achieved in this line did not interfere with cell division (metadata provided in Supplementary data 3).

    The authors should provide data concerning the efficiency of expression of the distinct markers after electroporation. This is provided in Supplementary Figure S1 (FACS data) and detailed above for this reviewer.

    *At Fig 1C, the schematic representation describes clone selection, however in the methods it is stated (L348-349): "Sorted cells expressing medium levels of fluorescence were expanded and frozen then representative lots of each polyclonal cell line...". There is some confusion regarding which experiments were performed using polyclonal medium-level mixed populations or monoclonal populations. *

    We apologise for any confusion and have revised the Figure 1C schematic to indicate that cells can be selected to either make polyclonal lines or clonal lines.

    *Reviewer #2 (Significance (Required)):

    The study provides novel tools, as well as elements regarding neuroepithelium biology. It is well conducted and written, and the quality of images is excellent. It reads more as a resource paper in its current version, since the observation regarding neural cell division and delamination are interesting but not deeply explored, so this review will focus on those technical aspects rather than the novelty of the biological findings.

    This study would be of interests for researchers in stem cells and organoids, developmental biology, and neurosciences.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In the manuscript, "Engineering fluorescent reporters in human pluripotent cells and strategies for live imaging human neurogenesis" the authors Dady et al. describe the adaptation of a recent advancement in transposase technology (HyPBase) as a method to integrate live reporters in human pluripotent stem cells. They show that these florescent reporters paired with new imaging strategies can be used to confirm the existence cellular behaviour described in other species such as the interkinetic nuclear migration (IKNM) of dividing progenitors in neural tube development. Finally, they demonstrate that this live imaging system is also able to discover novel biology by identifying previously undescribed actin polymerization at the basal nuclear surface of cortical progenitors undergoing cell division. Overall, the study presents two examples in which this adapted tool will aid in live-imaging studies of cellular biology.

    Major Concerns:

    1. This work needs more controls to properly demonstrate claims that their engineering strategy provides an advancement to current Piggyback methods. Their HyPBase strategy needs to be compared and quantified in terms of efficiency with other methods to support their claims (increased detection and reduced phototoxicity).*

    We do not make specific claims for our experiments with respect to the superiority of HyPBase strategy. Our comments on this approach referred to by the reviewer here are in the Introduction (L 94-103), are supported by the literature (e.g. more stable gene expression than native piggyBac or the Tc1/mariner transposase Sleeping Beauty (Doherty, et al., 2012, Yusa, et al., 2011) and serve to explain our selection of HyPBase for our experiments. We make a case for using HyPBase as opposed to another transposase and although it would be interesting to compare efficiencies, this comment does not specify what “other methods” might be informative.

    2.Throughout the manuscript more quantification is needed of the results. How many rosettes were examined? Were all the reported cells within one rosette? Were there differences between rosettes? This should be done for both the spinal and cortical differentiations.

    The reviewer appears to have missed this information – we placed detailed quantifications in the figure legends (numbers of independent experiments and rosettes) and in the Methods in a specific section on Quantification of cell behaviour (L465-486), rather than in the main text. These has since been further updated and we now also provide additional metadata in the form of Excel spreadsheets for quantifications and analyses made for both spinal cord and cortical rosettes (Supplementary data 3 and 4 respectively).

    *Minor Comments:

    1. Line 246 needs quantification shown in figures of the statements made. Specifically, how many cells were measured to get this number?*

    This information was provided in the figure 4 legend and we have since added numbers to these data; we were able to monitor 169 divisions in 21 rosettes; 154/166 divisions had vertical cleavage planes (symmetric) and 12/166 had horizontal cleavage planes (asymmetric).

    These detailed observations were made in two independent experiments, along with observations of basal nuclear membrane F-Tractin localisation. This is noted in figure 4 legend, Methods and detailed metadata is provided in Supplementary data 4.

    2.How many cells in the cortical rosettes had the enriched actin at the basal nuclear surface?

    We confidently observed basal nuclear membrane F-Tractin enrichment in 141/146 divisions, for the remaining 20 cases (166-146), we could not tell whether F-Tractin is enriched or not at the basal nuclear membrane either because of low expression levels or because the basal nuclear membrane was out of focus at NEB. In 5 cases, we did not see the basal nuclear enrichment despite sufficient F-Tractin expression levels and the nucleus being in focus. We have updated the Fig4 legend excluding the non-analysable cases and see detailed metadata is provided in Supplementary data 4.

    *Reviewer #3 (Significance (Required)):

    General Assessment: This manuscript makes a very minor advancement in the field of stem cell engineering and developmental biology, but one that is worthy of publication with a few edits.

    Advance: While PiggyBac reporters are widely used in stem cell engineering, Dady et al. demonstrate a new workflow using HyPBase which would be beneficial to the field. However, to increase this benefit, much more description and quantification of the methods would be needed. The biological advances of this manuscript are also very minor, but interesting as most of them confirm that human neural rosettes mimic many of the observed cell behaviours seen in animal models. Along these lines is the actin dynamics observation in cortical rosettes is interesting, but a preliminary observation and in need of follow up experiments.

    Audience: Regardless, this technique would be of interest to the wider field of stem cell engineering.

    My Expertise: Human Stem Cell Engineering, Neural Tube Development*

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    In the manuscript, "Engineering fluorescent reporters in human pluripotent cells and strategies for live imaging human neurogenesis" the authors Dady et al. describe the adaptation of a recent advancement in transposase technology (HyPBase) as a method to integrate live reporters in human pluripotent stem cells. They show that these florescent reporters paired with new imaging strategies can be used to confirm the existence cellular behaviour described in other species such as the interkinetic nuclear migration (IKNM) of dividing progenitors in neural tube development. Finally, they demonstrate that this live imaging system is also able to discover novel biology by identifying previously undescribed actin polymerization at the basal nuclear surface of cortical progenitors undergoing cell division. Overall, the study presents two examples in which this adapted tool will aid in live-imaging studies of cellular biology.

    Major Concerns:

    1.This work needs more controls to properly demonstrate claims that their engineering strategy provides an advancement to current Piggyback methods. Their HyPBase strategy needs to be compared and quantified in terms of efficiency with other methods to support their claims (increased detection and reduced phototoxicity). 2.Throughout the manuscript more quantification is needed of the results. How many rosettes were examined? Were all the reported cells within one rosette? Were there differences between rosettes? This should be done for both the spinal and cortical differentiations

    Minor Comments:

    1.Line 246 needs quantification shown in figures of the statements made. Specifically how many cells were measured to get this number? 2.How many cells in the cortical rosettes had the enriched actin at the basal nuclear surface?

    Significance

    General Assessment: This manuscript makes a very minor advancement in the field of stem cell engineering and developmental biology, but one that is worthy of publication with a few edits.

    Advance: While PiggyBac reporters are widely used in stem cell engineering, Dady et al. demonstrate a new workflow using HyPBase which would be beneficial to the field. However, to increase this benefit, much more description and quantification of the methods would be needed. The biological advances of this manuscript are also very minor, but interesting as most of them confirm that human neural rosettes mimic many of the observed cell behaviours seen in animal models. Along these lines is the actin dynamics observation in cortical rosettes is interesting, but a preliminary observation and in need of follow up experiments.

    Audience: Regardless, this technique would be of interest to the wider field of stem cell engineering.

    My Expertise: Human Stem Cell Engineering, Neural Tube Development

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript describes the generation of novel lines of human pluripotent stem cells bearing fluorescent reporters, engineered through piggyBac transposon-mediated integration. The cells are differentiated into neuronal organoids, allowing to capture cellular behaviors associated to cell division. A replating protocol allows the observation of aging neurons by reducing the thickness of the tissue thereby facilitating live imaging. The authors also leverage the transposon technology to create mosaically-labelled organoids which allows visualizing aspects of neuronal delamination, notably cytoskeleton dynamics. They discover an undescribed pattern of F-actin enrichment at the basal nuclear membrane prior to nuclear envelope breakdown.

    L104-109: "Moreover, the transposon system obviates drawbacks of directly engineering endogenous proteins...". Despite the risk of endogenous protein dysfunction, directly tagging allows the full regulation of gene expression (including the promoter, the enhancers and other regulatory regions rather than a strong constitutive promoter such as CAG). In addition, the number of copies integrated and the genomic regions are variable with PB, which does not reflect the endogenous expression. This could be rephrased by nuancing the advantages and drawbacks of each approach. The PiggyBac method is easier and faster, but it results in overexpression of a tagged protein that will be expressed since the hESC state and might not reflect the expression dynamics of the endogenous protein.

    L124-126: "To monitor cell shape and dynamics we used a plasma membrane (pm) localized protein tagged with eGFP or mKate2 (pm-eGFP or pm-mKate2)." Could the authors provide more details and a reference on the palmitoylated rat peptide use to force membrane expression?

    L132-133: " Finally, to observe actin cytoskeletal dynamics we selected F-tractin, for its minimal impact on cytoskeletal homeostasis..". A recent JCB paper (https://doi.org/10.1083/jcb.202409192) suggests that "F-tractin alters actin organization and impairs cell migration when expressed at high levels". Whether the overexpression of F-tractin in hESC using a CAG promoter reflects the physiological F-actin dynamics and/or if the high levels could lead to an alteration of cell behavior should be addressed or at least discussed.

    L146-147: "...to generate polyclonal cell lines selected for expression of easily detectable (medium level) fluorescence for live imaging studies". What are the criteria used to define medium level? Number of copies integrated into the genome? Or levels by FACS during clone selection?

    L260-263: "Efficient stable integration and moderate expression levels were achieved by optimising, i) the quantity and ratio of piggyBac plasmids and transposase and ii) subsequent FACS to exclude high expressing cells, as well as iii) transfection methods, including temporally defined lipofection in hiPSC-derived tissues." The ration 5:1 is classically used for PB Transposase delivery, however there is still high variability in the number of copies integration. Lipofection in derived tissues has been shown to be challenging. Could the authors should provide quantitative data regarding the efficiency of their approaches, notably the level of mosaicism one could expect?

    L191-194: "We further wished to monitor sub-cellular behaviour within the developing neuroepithelium. To achieve this, we devised a strategy to target a mosaic of cells in established neural rosettes using lipofection. PiggyBac constructs and HyPBase transposase were transfected into D8/D9 human spinal cord neural progenitors using lipofectamine (Felgner, et al., 1987)(Fig. 3A)." The mosaicism is not an all or nothing in this method but also leads to variations in expression levels among the positive cells. The protocol for lipofection could be better detailed to allow easy reproduction by other teams, and its expected efficiency should be discussed. It would be interesting to explore the relationship between individual cells phenotype and expression levels.

    Additional comments:

    • Did the authors perform karyotyping of the hPSCs prior to use in the differentiation protocol?
    • Were pluripotency assays performed after reporter lines generation?
    • Did the authors measure the cell proliferation rate in H2B-overexpressing cells and controls? Since H2B plays an important role in cytokinesis, it could interfere in cell division when H2B is overexpressed (see doi: 10.3390/cells8111391). The authors should provide data concerning the efficiency of expression of the distinct markers after electroporation. At Fig 1C, the schematic representation describes clone selection, however in the methods it is stated (L348-349): "Sorted cells expressing medium levels of fluorescence were expanded and frozen then representative lots of each polyclonal cell line...". There is some confusion regarding which experiments were performed using polyclonal medium-level mixed populations or monoclonal populations.

    Significance

    The study provides novel tools, as well as elements regarding neuroepithelium biology. It is well conducted and written, and the quality of images is excellent. It reads more as a resource paper in its current version, since the observation regarding neural cell division and delamination are interesting but not deeply explored, so this review will focus on those technical aspects rather than the novelty of the biological findings.

    This study would be of interests for researchers in stem cells and organoids, developmental biology, and neurosciences.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Dady et al have developed fluorescent reporters to enable live imaging of cell behaviour and morphology in human pluripotent stem cell lines (PSCs). These reporters target 3 main features, the plasma membrane, nucleus and cytoskeleton. Reporter PSCs have been generated using a piggyBac transposon-mediated stable integration strategy, using a hyperactive piggyBac transposase (HyPBase). The same constructs were also used for mosaic labelling of cells within 2D cultures using lipofectamine transfection.

    The reporters used are tagged with either eGFP or mKate2 (far red) and tag the plasma membrane (pm) via the addition of a 20 amino-acid sequence from rat GAP-43 to the N-terminus of the fluorescent protein, the nucleus via Histone 2B with a laser-mediated photo-conversion option (H2B-mEos3.2), and the cytoskeleton via F-Tractin. In total, the authors produced lines with the following:

    • pm-mKate2 (far red)
    • pm-eGFP (green)
    • H2B-mEos3.2 (green to red)
    • F-tractin-mKate2 (far red)
    • H2B-mEos3.2 and pm-mKate2 (green to red, plus far red)

    The cell lines used to generate these were the human embryonic stem cell line H9 and human induced pluripotent cell line ChiPS4. The constructs were also used to label cells in a mosaic fashion, using lipofectamine transfection of the original cell lines once they had formed neural rosettes.

    Using these cells, Dady et al then performed live imaging in vitro of human spinal cord rosettes and assessed cell behaviour. In particular they analysed mitotic cleavage planes and apical positioning of neural progenitor cells (NPCs), and assessed actin dynamics within these cells. They showed a slowing of the cell cycle length after the initial expansion phase, an increase in the rate of asymmetric division of these NPCs, and abscission of the apical membrane during these divisions. The F-tractin reporter showed enrichment at the basal nuclear membrane during these cell divisions, suggested to help prevent basal chromosome displacement during mitosis.

    Major comments:

    The data presented are convincing and could be strengthened by the following additions and clarifications:

    1. How long do the fluorescent reports take to be visible when transfected via lipofectamine? How efficiently are they expressed? And what concentrations were tested to enable the mosaic expression presented?
    2. Will these cell lines and constructs be made publicly available after publication?
    3. Were the H9 and ChiPS4 lines characterised after the reporters were added to show they still proliferate/differentiate as they did prior to the reporter integration?
    4. Can the novel actin dynamics described be quantified? How many cells imaged show these novel dynamics?

    Minor comments:

    1. Some images in the figures and supplemental movies are low in resolution, for example the DAPI in Fig 4B, making it hard to distinguish individual cells. Please increase this.
    2. Please show a merge of Phallodin and F-Tractin in Fig4, this will help the colocalization to be fully appreciated.
    3. Some additional annotation on the supplemental movies would be useful to indicate to the reader exactly what cell to follow.

    Significance

    Human neurogenesis is currently poorly understood compared to many model systems used, yet key differences have already been identified between the human and the mouse, prompting the need for further investigation of human neural development. A major reason that human neurogenesis has been difficult to study is a lack of tools to enable cell morphology and behaviours to be analysed in real time.

    The reporters and reporter PSC lines generated by Dady et al will allow many of these cell characteristics to be observed using live imaging. For example, the morphology of neural progenitors during and after cell divisions, how the apical and basal processes and membranes are divided, and how the actin cytoskeleton helps to regulate these processes.

    Importantly, PSC lines can be very heterogeneous, making generating reporter lines costly and time intensive. The use of these reporters with lipofectamine transfection, for a mosaic labelling, allows the visualisation of the plasma membrane, nucleus and cytoskeleton in any human PSC/NPC line, or even in human tissue cultures, without the need to generate each specific reporter line, making it a valuable tool for many labs in the field.

  5. Version published to 10.1101/2024.05.01.591467 on bioRxiv