Programming human cell fate: overcoming challenges and unlocking potential through technological breakthroughs.

In recent years, there have been notable advancements in the ability to programme human cell identity, enabling us to design and manipulate cell function in a Petri dish. However, current protocols for generating target cell types often lack efficiency and precision, resulting in engineered cells that do not fully replicate the desired identity or functional output. This applies to different methods of cell programming, which face similar challenges that hinder progress and delay the achievement of a more favourable outcome. However, recent technological and analytical breakthroughs have provided us with unprecedented opportunities to advance the way we programme cell fate. The Company of Biologists' 2023 workshop on 'Novel Technologies for Programming Human Cell Fate' brought together experts in human cell fate engineering and experts in single-cell genomics, manipulation and characterisation of cells on a single (sub)cellular level. Here, we summarise the main points that emerged during the workshop's themed discussions. Furthermore, we provide specific examples highlighting the current state of the field as well as its trajectory, offering insights into the potential outcomes resulting from the application of these breakthrough technologies in precisely engineering the identity and function of clinically valuable human cells.

Immune cells in cardiac repair and regeneration.

The immune system is fundamental to tissue homeostasis and is the first line of defense following infection, injury or disease. In the damaged heart, large numbers of immune cells are recruited to the site of injury. These cells play an integral part in both repair by scar formation and the initiation of tissue regeneration. They initially assume inflammatory phenotypes, releasing pro-inflammatory cytokines and removing dead and dying tissue, before entering a reparative stage, replacing dead muscle tissue with a non-contractile scar. In this Review, we present an overview of the innate and adaptive immune response to heart injury. We explore the kinetics of immune cell mobilization following cardiac injury and how the different innate and adaptive immune cells interact with one another and with the damaged tissue. We draw on key findings from regenerative models, providing insight into how to support a robust immune response permissible for cardiac regeneration. Finally, we consider how the latest technological developments can offer opportunities for a deeper and unbiased functional understanding of the immune response to heart disease, highlighting the importance of such knowledge as the basis for promoting regeneration following cardiac injury in human patients.

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.

Prrx1b restricts fibrosis and promotes Nrg1-dependent cardiomyocyte proliferation during zebrafish heart regeneration.

Fibroblasts are activated to repair the heart following injury. Fibroblast activation in the mammalian heart leads to a permanent fibrotic scar that impairs cardiac function. In other organisms, such as zebrafish, cardiac injury is followed by transient fibrosis and scar-free regeneration. The mechanisms that drive scarring versus scar-free regeneration are not well understood. Here, we show that the homeobox-containing transcription factor Prrx1b is required for scar-free regeneration of the zebrafish heart as the loss of Prrx1b results in excessive fibrosis and impaired cardiomyocyte proliferation. Through lineage tracing and single-cell RNA sequencing, we find that Prrx1b is activated in epicardial-derived cells where it restricts TGFß ligand expression and collagen production. Furthermore, through combined in vitro experiments in human fetal epicardial-derived cells and in vivo rescue experiments in zebrafish, we conclude that Prrx1 stimulates Nrg1 expression and promotes cardiomyocyte proliferation. Collectively, these results indicate that Prrx1 is a key transcription factor that balances fibrosis and regeneration in the injured zebrafish heart. This article has an associated 'The people behind the papers' interview.

The ontogeny, activation and function of the epicardium during heart development and regeneration.

The epicardium plays a key role during cardiac development, homeostasis and repair, and has thus emerged as a potential target in the treatment of cardiovascular disease. However, therapeutically manipulating the epicardium and epicardium-derived cells (EPDCs) requires insights into their developmental origin and the mechanisms driving their activation, recruitment and contribution to both the embryonic and adult injured heart. In recent years, studies of various model systems have provided us with a deeper understanding of the microenvironment in which EPDCs reside and emerge into, of the crosstalk between the multitude of cardiovascular cell types that influence the epicardium, and of the genetic programmes that orchestrate epicardial cell behaviour. Here, we review these discoveries and discuss how technological advances could further enhance our knowledge of epicardium-based repair mechanisms and ultimately influence potential therapeutic outcomes in cardiovascular regenerative medicine.

Impact of KLF4 on Cell Proliferation and Epithelial Differentiation in the Context of Cystic Fibrosis.

Cystic fibrosis (CF) cells display a more cancer-like phenotype vs. non-CF cells. KLF4 overexpression has been described in CF and this transcriptional factor acts as a negative regulator of wt-CFTR. KLF4 is described as exerting its effects in a cell-context-dependent fashion, but it is generally considered a major regulator of proliferation, differentiation, and wound healing, all the processes that are also altered in CF. Therefore, it is relevant to characterize the differential role of KLF4 in these processes in CF vs. non-CF cells. To this end, we used wt- and F508del-CFTR CFBE cells and their respective KLF4 knockout (KO) counterparts to evaluate processes like cell proliferation, polarization, and wound healing, as well as to compare the expression of several epithelial differentiation markers. Our data indicate no major impact of KLF4 KO in proliferation and a differential impact of KLF4 KO in transepithelial electrical resistance (TEER) acquisition and wound healing in wt- vs. F508del-CFTR cells. In parallel, we also observed a differential impact on the levels of some differentiation markers and epithelial-mesencymal transition (EMT)-associated transcription factors. In conclusion, KLF4 impacts TEER acquisition, wound healing, and the expression of differentiation markers in a way that is partially dependent on the CFTR-status of the cell.

CFTR supports cell death through ROS-dependent activation of TMEM16F (anoctamin 6).

Cystic fibrosis transmembrane conductance regulator (CFTR) is the essential chloride and bicarbonate channel in the apical membrane of epithelial cells. CFTR was also proposed earlier to conduct glutathione (GSH) out of airway epithelial cells to be enriched in the apical airway surface liquid to neutralize reactive oxygen species (ROS). Although earlier studies suggested that release of GSH by wild type (wt) CFTR may lead to an increase in cytosolic ROS, we did not detect different ROS levels in cells expressing wt-CFTR and mutant F508del-CFTR, independent of CFTR-activation or exposure to the ROS donor tert-butyl hydroperoxide. The Ca2+-activated phospholipid scramblase and ion channel TMEM16F (anoctamin 6, ANO6) is also expressed in airway cells. ANO6 produced outwardly rectifying Cl- currents (ORCC) and scrambled plasma membrane phospholipids when activated by increase in cytosolic ROS and consecutive peroxidation of plasma membrane lipids. ANO6 activity is enhanced by CFTR, probably through translocation of signaling proteins to the plasma membrane. The present data suggest that enhanced cell death in CFTR-expressing cells is due to upregulation of ANO6-activity. In ANO6 knockout mice, the number of apoptotic cells in the intestinal epithelium was strongly reduced, supporting the role of ANO6 for cell death. Thus, ANO6 and CFTR act cooperatively on ROS-mediated cell death, which is not further augmented by cAMP-dependent stimulation. We propose that ANO6 supports cell death correlated with expression of CFTR, possibly by inducing ferroptosis.

A novel TGFß modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

As some of the most widely utilised intercellular signalling molecules, transforming growth factor ß (TGFß) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFß signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFß signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFß signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFß signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFß signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFß family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFß signalling and thereby embryonic patterning.

Elemental Quantification and Residues Characterization of Wet Digested Certified and Commercial Carbon Materials.

Inductively coupled plasma optical emission spectroscopy (ICP-OES) is a common, relatively low cost, and straightforward analytical technique for the study of trace quantities of metals in solid materials, but its applicability to nanocarbons (e.g., graphene and nanotubes) has suffered from the lack of efficient digestion steps and certified reference materials (CRM). Here, various commercial and certified graphitic carbon materials were subjected to a "two-step" microwave-assisted acid digestion procedure, and the concentrations of up to 18 elements were analyzed by ICP-OES. With one exception (Sm), successful quantification of all certified elements in the two reference nanocarbons studied was achieved, hence validating the sample preparation approach used. The applicability of our "two-step" protocol was further confirmed for a commercial single-walled carbon nanotube sample. However, the digestion was markedly incomplete for all other commercial materials tested. Where possible, the digestion residues of the carbon materials analyzed (CRM included) were characterized to understand the structural changes that take place and how this may explain the challenge of disintegrating graphitic carbon. In this respect, it was found that solid state nuclear magnetic resonance holds considerable promise as a nonlocalized, easily interpretable, and reliable tool to access the efficient disintegration of these materials.

Fgf differentially controls cross-antagonism between cardiac and haemangioblast regulators.

Fibroblast growth factor (Fgf) has been implicated in the control of heart size during development, although whether this is by controlling cell fate, survival or proliferation has not been clear. Here, we show that Fgf, without affecting survival or proliferation, acts during gastrulation to drive cardiac fate and restrict anterior haemangioblast fate in zebrafish embryos. The haemangioblast programme was thought to be activated before the cardiac programme and is repressive towards it, suggesting that activation by Fgf of the cardiac programme might be via suppression of the haemangioblast programme. However, we show that the cardiac regulator nkx2.5 can also repress the haemangioblast programme and, furthermore, that cardiac specification still requires Fgf signalling even when haemangioblast regulators are independently suppressed. We further show that nkx2.5 and the cloche candidate gene lycat are expressed during gastrulation and regulated by Fgf, and that nkx2.5 overexpression, together with loss of the lycat targets etsrp and scl can stably induce expansion of the heart. We conclude that Fgf controls cardiac and haemangioblast fates by the simultaneous regulation of haemangioblast and cardiac regulators. We propose that elevation of Fgf signalling in the anterior haemangioblast territory could have led to its recruitment into the heart field during evolution, increasing the size of the heart.

Distinct epicardial gene regulatory programs drive development and regeneration of the zebrafish heart.

Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart fully regenerates following injury. Reactivation of cardiac developmental programs is considered key to successfully regenerating the heart, yet the regulation underlying the response to injury remains elusive. Here, we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardia. We identified epicardial enhancer elements with specific activity during development or during adult heart regeneration. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programs driving each of these processes, which were largely distinct. Loss of Hif1ab, Nrf1, Tbx2b, and Zbtb7a, central regulators of the regenerating epicardial network, in injured hearts resulted in elevated epicardial cell numbers infiltrating the wound and excess fibrosis after cryoinjury. Our work identifies differences between the regulatory blueprint deployed during epicardial development and regeneration, underlining that heart regeneration goes beyond the reactivation of developmental programs.

Cardiopoietic factors: extracellular signals for cardiac lineage commitment.

Cardiac muscle creation during embryogenesis requires extracellular instructive signals that are regulated precisely in time and space, intersecting with intracellular genetic programs that confer or fashion the ability of the cells to respond. Unmasking the essential signals for cardiac lineage decisions has paramount importance for cardiac development and regenerative medicine, including the directed differentiation of progenitor and stem cells to a cardiac muscle fate.

Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in zebrafish.

Due to its genetic amenability coupled with advances in genome editing, zebrafish is an excellent model to examine the function of (epi)genomic elements. Here, we repurposed the Ac/Ds maize transposition system to efficiently characterise zebrafish cis-regulated elements, also known as enhancers, in F0-microinjected embryos. We further used the system to stably express guide RNAs enabling CRISPR/dCas9-interference (CRISPRi) perturbation of enhancers without disrupting the underlying genetic sequence. In addition, we probed the phenomenon of antisense transcription at two neural crest gene loci. Our study highlights the utility of Ac/Ds transposition as a new tool for transient epigenome modulation in zebrafish.

Modelling the pathology and treatment of cardiac fibrosis in vascularised atrial and ventricular cardiac microtissues.

Introduction: Recent advances in human cardiac 3D approaches have yielded progressively more complex and physiologically relevant culture systems. However, their application in the study of complex pathological processes, such as inflammation and fibrosis, and their utility as models for drug development have been thus far limited. Methods: In this work, we report the development of chamber-specific, vascularised human induced pluripotent stem cell-derived cardiac microtissues, which allow for the multi-parametric assessment of cardiac fibrosis. Results: We demonstrate the generation of a robust vascular system in the microtissues composed of endothelial cells, fibroblasts and atrial or ventricular cardiomyocytes that exhibit gene expression signatures, architectural, and electrophysiological resemblance to in vivo-derived anatomical cardiac tissues. Following pro-fibrotic stimulation using TGFß, cardiac microtissues recapitulated hallmarks of cardiac fibrosis, including myofibroblast activation and collagen deposition. A study of Ca2+ dynamics in fibrotic microtissues using optical mapping revealed prolonged Ca2+ decay, reflecting cardiomyocyte dysfunction, which is linked to the severity of fibrosis. This phenotype could be reversed by TGFß receptor inhibition or by using the BET bromodomain inhibitor, JQ1. Discussion: In conclusion, we present a novel methodology for the generation of chamber-specific cardiac microtissues that is highly scalable and allows for the multi-parametric assessment of cardiac remodelling and pharmacological screening.

Cross-talk of inflammatory mediators and airway epithelium reveals the cystic fibrosis transmembrane conductance regulator as a major target.

Airway inflammation, mucus hyperproduction and epithelial remodelling are hallmarks of many chronic airway diseases, including asthma, COPD and cystic fibrosis. While several cytokines are dysregulated in these diseases, most studies focus on the response of airways to interleukin (IL)-4 and IL-13, which have been shown to induce mucus hyperproduction and shift the airway epithelium towards a hypersecretory phenotype. We hypothesised that other cytokines might induce the expression of chloride (Cl-) channels/transporters, and regulate epithelial differentiation and mucus production. To this end, fully differentiated human airway basal cells (BCi-NS1.1) were treated with cytokines identified as dysregulated in those diseases, namely IL-8, IL-1ß, IL-4, IL-17A, IL-10 and IL-22, and tumour necrosis factor-α. Our results show that the cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl- channel modulated by inflammation, in contrast to transmembrane protein 16A (TMEM16A), whose levels only changed with IL-4. Furthermore, we identified novel roles for IL-10 and IL-22 by influencing epithelial differentiation towards ciliated cells and away from pulmonary ionocytes. In contrast, IL-1ß and IL-4 reduced the number of ciliated cells while increasing club cells. Interestingly, while IL-1ß, IL-4 and IL-10 upregulated CFTR expression, IL-4 was the only cytokine that increased both its function and the number of CFTR-expressing club cells, suggesting that this cell type may be the main contributor for CFTR function. Additionally, all cytokines assessed increased mucus production through a differential upregulation of MUC5AC and MUC5B transcript levels. This study reveals a novel insight into differentiation resulting from the cross-talk of inflammatory mediators and airway epithelial cells, which is particularly relevant for chronic airway diseases.

Functional Heterogeneity within the Developing Zebrafish Epicardium.

The epicardium is essential during cardiac development, homeostasis, and repair, and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity, and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA sequencing uncovered three epicardial subpopulations with specific genetic programs and distinctive spatial distribution. Perturbation of unique gene signatures uncovered specific functions associated with each subpopulation and established epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. Understanding which mechanisms epicardial cells employ to establish a functional epicardium and how they communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease.

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.

Chemical and Structural Analysis of Carbon Materials Subjected to Alkaline Oxidation.

Redox species such as transition metals may, unknowingly, integrate carbon materials that are produced (or supplied) for the assembling of electrodes in batteries, supercapacitors, and fuel cells. The extent to which these species alter the electrochemical profile of carbons and affect the performance and/or degradation of energy storage systems is still not fully appreciated. Alkaline oxidation (or fusion) is a promising approach to disintegrate nanocarbons for the subsequent study of their chemical composition by routine analytical tools. In this work, three commercial carbon powders, relevant for electrochemical applications and bearing varied textural orientation (point, radial, and planar), were selected to evaluate the versatility of fusion as a pretreatment process for elemental analysis. Additionally, the interaction of the flux, a lithium borate salt, with the carbons was elucidated by examining their post-fusion residues. The degree of structural degradation varied and, generally, the doping with Li and/or B (whether substitutional or interstitial) was low to nonexistent. With future developments, fusion could become a relevant pretreatment method to analyze the composition of carbon materials, even when complex mixtures (e.g., cycled battery electrodes) and larger batch scales are considered.

TMEM16A chloride channel does not drive mucus production.

Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.