Fate Decisions Research Articles

Abstract 2014: Long Non-coding RNA LINC-PINT Regulates Smooth Muscle Cell Plasticity In Developing Abdominal Aortic Aneurysms

Background: Long noncoding RNAs (lncRNAs) play pivotal roles as molecular regulators in diverse vascular biological processes and diseases. This study utilized multi-transcriptomic profiling to investigate the contribution of lncRNAs in Abdominal Aortic Aneurysm (AAA) disease. Materials & Methods: Utilizing bulk and single-cell RNA sequencing, we identified lncRNA long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT) as a significantly deregulated transcript in AAA compared to non-dilated aortas. Deconvolution of single-cell and bulk RNA-sequencing via the BayesPrism tool identified plasticity of smooth muscle cells (SMCs) as the predominant shifted condition between dilated vs. non-dilated aortas. Hybridization-based RNA in situ sequencing (HybRISS) confirmed the presence of LINC-PINT in de-differentiating SMCs. Results: Experimental knock-down of LINC-PINT , using site-specific antisense oligonucleotides (LNA-GapmeRs) markedly decreased proliferation rates of cultured human aortic smooth muscle cells (AoSMCs), but also increased their apoptosis, whereas overexpression of LINC-PINT had the opposite effect. Transcriptomic profiling of LINC-PINT -silenced AoSMCs indicated down-regulation of contractility markers, accompanied by an increase of inflammation and extracellular matrix (ECM) degradation. In line with this, LINC-PINT knock-down abolished the transforming growth factor beta (TGF-β)- induced AoSMCs polarization towards a contractile phenotype. Notably, RNA immunoprecipitation (RIP) demonstrated an interaction between LINC-PINT and p65, inhibiting its translocation to the promoter region of inflammatory genes. LINC-PINT knock-down increased p65 binding to its coactivator p50, which was confirmed by Co-immunoprecipitation (CO-IP). Combined in silico prediction and luciferase reporter assays further revealed that LINC-PINT expression was controlled by the ZNF93 and ZNF263 transcription factors. Conclusion: We provide evidence of LINC-PINT being a novel regulator influencing SMC dynamics and fate decisions of central importance to AAA pathophysiology. Currently ongoing in vivo studies in Lncpint -deficient mice explore its relevance in experimental AAA development.

Deciphering the role of Klf2 in CD8 T cell differentiation and exhaustion during chronic infection

Abstract T cell exhaustion, a state of dysfunction occurring during chronic infections and cancer, is marked by a progressive loss of effector functions and increased expression of inhibitory receptors. In chronic infection, CD8 T cells differentiate into TCF1+ progenitor (TPRO), CX3CR1+ effector (TEFF), and terminally exhausted (TEXH) subsets. The differentiation pathways are governed by complex transcription factor (TF) networks. Employing regulon analysis enabled by single-cell RNA+ATAC multiome sequencing, we dissected the TF networks orchestrating the fate of exhausted CD8 T cell populations. Our integrative analysis, complemented by single-cell CRISPR screening, highlighted Krüppel-like factor 2 (KLF2) as a pivotal TF necessary for maintaining TPRO cells and generating TEFF cells. Our research also revealed that KLF2 regulates the distinct distribution patterns of CD8 T cell subsets across organs. More intriguingly, multiomic analysis uncovered a novel function of Klf2 in limiting T cell exhaustion. Klf2-deficient CD8 T cells exhibited a marked increase in exhaustion signature genes. ATAC-seq and CUT&TAG assays further revealed different chromatin accessibility and histone modifications within Klf2 and its enhancers across CD8 T cell subsets. These insights underscore the differential transcriptional and epigenetic regulation of Klf2, presenting a novel mechanism that influences the cell fate decision of CD8 T cells during chronic infections.

Elucidating inflammation-induced cell fate decisions in primary human Tregs

Abstract Adoptive regulatory T-cell (Treg) therapy has remarkable efficacy in promoting immune tolerance in preclinical models of transplantation and autoimmune disease. However, lineage-tracing studies have revealed that Tregs can undergo epigenetic reprogramming in chronically inflamed tissue environments, resulting in the acquisition of proinflammatory functions and the capacity to exacerbate autoimmunity. Despite intense interests in developing Treg therapy for humans, inflammation-induced human Treg lineage-decommitment remains poorly understood. Here, we present a robust in vitro model of IL6, IL1β, and IL23-driven human Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3 conserved non-coding sequence (CNS)-2 enhancer re-methylation, diminished in vitro suppressive function, and elevated proinflammatory cytokine expression. Single-cell transcriptomic and chromatin accessibility analyses of human Tregs undergoing destabilization revealed a gradual closing of a Treg-specific element (A) and a reciprocal opening of a competing element (B) at the IRF4 locus. CRISPR-mediated excision of the B element (IRF4ΔB) preserved Treg identity and enhanced in vitro suppressive activity following prolonged inflammatory exposure. Ongoing experiments aim to evaluate the in vivo function and stability of IRF4ΔB Tregs in a xenogeneic graft-versus-host model. These findings may facilitate the design of Treg therapeutics with greater potency and improved safety.

NAT10-mediated ac4C modification promotes stemness and chemoresistance of colon cancer by stabilizing NANOGP8

BackgroundColon cancer (CC) stem cells can self-renew as well as expand, thereby promoting tumor progression and conferring resistance to chemotherapeutic agents. The acetyltransferase NAT10 mediates N4-acetylcytidine (ac4C) modification, which in turn drives tumorigenesis, metastasis, stemness properties maintenance, and cell fate decisions. Nonetheless, the specific involvement of ac4C modification mediated by NAT10 in regulating stemness and chemosensitivity in CC remains undetermined. MethodsThe levels of NAT10 in normal colon and chemoresistant CC tissues were determined utilizing quantitative real-time polymerase chain reaction alongside immunohistochemistry. Assessing cancer cell stemness and chemosensitivity was conducted by various methods including spheroid and colony formation, western blotting, and flow cytometry. RNA-Seq was used to identify target genes, and RNA immunoprecipitation analysis was used to explore the potential mechanisms. ResultsWe observed NAT10 overexpression and increased ac4C modification levels in chemoresistant CC tissues. The in vivo and in vitro analysis findings suggested that NAT10 promoted CC cell stemness while suppressing their chemosensitivity. Conversely, Remodelin, a NAT10-specific inhibitor, enhanced CC cell chemosensitivity. Mechanistically, NAT10 increased the level of NANOGP8 ac4C modification and promoted NANOGP8 mRNA stability. ConclusionsNAT10 promotes the maintenance of stemness and chemoresistance in CC cells by augmenting the mRNA stability of NANOGP8. The inhibition of NAT10 via Remodelin improves chemotherapeutic efficacy and impedes CC progression.

A logic-incorporated gene regulatory network deciphers principles in cell fate decisions.

Organisms utilize gene regulatory networks (GRN) to make fate decisions, but the regulatory mechanisms of transcription factors (TF) in GRNs are exceedingly intricate. A longstanding question in this field is how these tangled interactions synergistically contribute to decision-making procedures. To comprehensively understand the role of regulatory logic in cell fate decisions, we constructed a logic-incorporated GRN model and examined its behavior under two distinct driving forces (noise-driven and signal-driven). Under the noise-driven mode, we distilled the relationship among fate bias, regulatory logic, and noise profile. Under the signal-driven mode, we bridged regulatory logic and progression-accuracy trade-off, and uncovered distinctive trajectories of reprogramming influenced by logic motifs. In differentiation, we characterized a special logic-dependent priming stage by the solution landscape. Finally, we applied our findings to decipher three biological instances: hematopoiesis, embryogenesis, and trans-differentiation. Orthogonal to the classical analysis of expression profile, we harnessed noise patterns to construct the GRN corresponding to fate transition. Our work presents a generalizable framework for top-down fate-decision studies and a practical approach to the taxonomy of cell fate decisions.

A logic-incorporated gene regulatory network deciphers principles in cell fate decisions

Organisms utilize gene regulatory networks (GRN) to make fate decisions, but the regulatory mechanisms of transcription factors (TF) in GRNs are exceedingly intricate. A longstanding question in this field is how these tangled interactions synergistically contribute to decision-making procedures. To comprehensively understand the role of regulatory logic in cell fate decisions, we constructed a logic-incorporated GRN model and examined its behavior under two distinct driving forces (noise-driven and signal-driven). Under the noise-driven mode, we distilled the relationship among fate bias, regulatory logic, and noise profile. Under the signal-driven mode, we bridged regulatory logic and progression-accuracy trade-off, and uncovered distinctive trajectories of reprogramming influenced by logic motifs. In differentiation, we characterized a special logic-dependent priming stage by the solution landscape. Finally, we applied our findings to decipher three biological instances: hematopoiesis, embryogenesis, and trans-differentiation. Orthogonal to the classical analysis of expression profile, we harnessed noise patterns to construct the GRN corresponding to fate transition. Our work presents a generalizable framework for top-down fate-decision studies and a practical approach to the taxonomy of cell fate decisions.

Influence of DNA-methylation at multiple stages of limb chondrogenesis

Precise regulation of gene expression is of utmost importance during cell fate specification. DNA methylation is a key epigenetic mechanism that plays a significant role in the regulation of cell fate by recruiting repression proteins or inhibiting the binding of transcription factors to DNA to regulate gene expression. Limb development is a well-established model for understanding cell fate decisions, and the formation of skeletal elements is coordinated through a sequence of events that control chondrogenesis spatiotemporally. It has been established that epigenetic control participates in cartilage maturation. However, further investigation is required to determine its role in the earliest stages of chondrocyte differentiation. This study investigates how the DNA methylation environment affects cell fate divergence during the early chondrogenic events. Our research has shown for the first time that inhibiting DNA methylation in interdigital tissue with 5-azacytidine results in the formation of an ectopic digit. This discovery suggested that DNA methylation dynamics could regulate the fate of cells between chondrogenesis and cell death during autopod development. Our in vitro findings indicate that DNA methylation at the early stages of chondrogenesis is integral in regulating condensation by controlling cell adhesion and proapoptotic genes. As a result, the dynamics of methylation and demethylation are crucial in governing chondrogenesis and cell death during different stages of limb chondrogenesis.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

Chromatin landscape instructs precise transcription factor regulome during embryonic lineage specification

Embryos, originating from fertilized eggs, undergo continuous cell division and differentiation, accompanied by dramatic changes in transcription, translation, and metabolism. Chromatin regulators, including transcription factors (TFs), play indispensable roles in regulating these processes. Recently, the trophoblast regulator TFAP2C was identified as crucial in initiating early cell fate decisions. However, Tfap2c transcripts persist in both the inner cell mass and trophectoderm of blastocysts, prompting inquiry into Tfap2c's function in post-lineage establishment. In this study, we delineate the dynamics of TFAP2C during the mouse peri-implantation stage and elucidate its collaboration with the key lineage regulators CDX2 and NANOG. Importantly, we propose that de novo formation of H3K9me3 in the extraembryonic ectoderm during implantation antagonizes TFAP2C binding to crucial developmental genes, thereby maintaining its lineage identity. Together, these results highlight the plasticity of the chromatin environment in designating the genomic binding of highly adaptable lineage-specific TFs and regulating embryonic cell fates.

Single-cell analysis identifies critical regulators of spermatogonial development and differentiation in cattle-yak bulls

Spermatogenesis is a continuous process in which functional sperm are produced through a series of mitotic and meiotic divisions and morphological changes in germ cells. The aberrant development and fate transitions of spermatogenic cells cause hybrid sterility in mammals. Cattle-yak, a hybrid animal between taurine cattle (Bos taurus) and yak (Bos grunniens), exhibits male-specific sterility due to spermatogenic failure. In the present study, we performed single-cell RNA sequencing analysis to identify differences in testicular cell composition and the developmental trajectory of spermatogenic cells between yak and cattle-yak. The composition and molecular signatures of spermatogonial subtypes were dramatically different between these 2 animals, and the expression of genes associated with stem cell maintenance, cell differentiation and meiotic entry was altered in cattle-yak, indicating the impairment of undifferentiated spermatogonial fate decisions. Cell communication analysis revealed that signaling within different spermatogenic cell subpopulations was weakened, and progenitor spermatogonia were unable to or delayed receiving and sending signals for transformation to the next stage in cattle-yak. Simultaneously, the communication between niche cells and germ cells was also abnormal. Collectively, we obtained the expression profiles of transcriptome signatures of different germ cells and testicular somatic cell populations at the single-cell level and identified critical regulators of spermatogonial differentiation and meiosis in yak and sterile cattle-yak. The findings of this study shed light on the genetic mechanisms that lead to hybrid sterility and speciation in bovid species.

Bisphenol-F and Bisphenol-S (BPF and BPS) Impair the Stemness of Neural Stem Cells and Neuronal Fate Decision in the Hippocampus Leading to Cognitive Dysfunctions.

Neurogenesis occurs throughout life in the hippocampus of the brain, and many environmental toxicants inhibit neural stem cell (NSC) function and neuronal generation. Bisphenol-A (BPA), an endocrine disrupter used for surface coating of plastic products causes injury in the developing and adult brain; thus, many countries have banned its usage in plastic consumer products. BPA analogs/alternatives such as bisphenol-F (BPF) and bisphenol-S (BPS) may also cause neurotoxicity; however, their effects on neurogenesis are still not known. We studied the effects of BPF and BPS exposure from gestational day 6 to postnatal day 21 on neurogenesis. We found that exposure to non-cytotoxic concentrations of BPF and BPS significantly decreased the number/size of neurospheres, BrdU+ (proliferating NSC marker)and MAP-2+ (neuronal marker) cells and GFAP+ astrocytes in the hippocampus NSC culture, suggesting reduced NSC stemness and self-renewal and neuronal differentiation and increased gliogenesis. These analogs also reduced the number of BrdU/Sox-2+, BrdU/Dcx+, and BrdU/NeuN+ co-labeled cells in the hippocampus of the rat brain, suggesting decreased NSC proliferation and impaired maturation of newborn neurons. BPF and BPS treatment increases BrdU/cleaved caspase-3+ cells and Bax-2 and cleaved caspase protein levels, leading to increased apoptosis in hippocampal NSCs. Transmission electron microscopy studies suggest that BPF and BPS also caused degeneration of neuronal myelin sheath, altered mitochondrial morphology, and reduced number of synapses in the hippocampus leading to altered cognitive functions. These results suggest that BPF and BPS exposure decreased the NSC pool, inhibited neurogenesis, induced apoptosis of NSCs, caused myelin degeneration/synapse degeneration, and impaired learning and memory in rats.

EPAS1 expression contributes to maintenance of the primordial follicle pool in the mouse ovary

Oxygen availability can have profound effects on cell fate decisions and survival, in part by regulating expression of hypoxia-inducible factors (HIFs). In the ovary, HIF expression has been characterised in granulosa cells, however, any requirement in oocytes remains relatively undefined. Here we developed a Hif2a/Epas1 germline-specific knockout mouse line in which females were fertile, however produced 40% fewer pups than controls. No defects in follicle development were detected, and quality of MII oocytes was normal, as per assessments of viability, intracellular reactive oxygen species, and spindle parameters. However, a significant diminishment of the primordial follicle pool was evident in cKO females that was attributed to accelerated follicle loss from postnatal day 6 onwards, potentially via disruption of the autophagy pathway. These data demonstrate the importance of HIF signalling in oocytes, particularly at the primordial follicle stage, and lend to the importance of controlling oxygen tension in the development of in vitro growth and maturation approaches for assisted reproduction.

SRRM2 splicing factor modulates cell fate in early development.

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

SP6 controls human cytotrophoblast fate decisions and trophoblast stem cell establishment by targeting MSX2 regulatory elements

The commitment and differentiation of human placental progenitor cytotrophoblast (CT) cells are crucial for a successful pregnancy, but the underlying mechanism remains poorly understood. Here, we identified the transcription factor (TF), specificity protein 6 (SP6), as a human species-specific trophoblast lineage TF expressed in human placental CT cells. Using pluripotent stem cells as a model, we demonstrated that SP6 controls CT generation and the establishment of trophoblast stem cells (TSCs) and identified msh homeobox 2 (MSX2) as the downstream effector in these events. Mechanistically, we showed that SP6 interacts with histone acetyltransferase P300 to alter the landscape of H3K27ac at targeted regulatory elements, thereby favoring transcriptional activation and facilitating CT cell fate decisions and TSC maintenance. Our results established SP6 as a regulator of the human trophoblast lineage and implied its role in placental development and the pathogenies of placental diseases.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract LB289: Single-cell CRISPR screens at scale to understand T cell biology

Abstract CRISPR screens have become the primary discovery engine in modern biology and are widely used to uncover novel targets in immuno-oncology. However, these genetic screens are usually coupled to rather simplistic read-outs such as cell fitness. In contrast, CROP-Seq (or “CRISPR Droplet” Sequencing) screens combine CRISPR perturbation with single-cell transcriptomics, enabling high-content phenotyping at single-cell resolution. Briefly, cells are perturbed with a pooled sgRNA library and transcriptomic profiles of each cell are recorded using conventional single-cell RNA sequencing platforms. Currently, the scale of these screens is limited to the perturbation of a couple of hundred genes, possibly up to 1.000 genes, because of increased costs for single-cell library preparation and next generation sequencing (NGS). At Myllia, we have built a platform allowing high-content CROP-Seq screens in both CRISPR-ready cancer cell lines as well as primary immune cells, e.g., human T cells. Using CRISPR KO, CRISPR interference or CRISPR activation workflows, we can help accelerate the identification of target genes and regulatory networks involved in immuno-oncology. Primary T cells are of great interest in the immunotherapy community as they are key players in autoimmune and inflammatory disease. Engineered T-lymphocytes such as CAR-T cells are currently developed as novel cellular medicines, yet many T cell-intrinsic features involved in CAR-T potency remain elusive. To enable the discovery of novel targets, we have performed multiple customized CROP-Seq CRISPR KO screens in primary human T cells investigating T cell stemness and effector phenotypes as well as CD4+ T helper cell differentiation towards Th1, Th2, or Th17 subsets. Here, we present screens for regulators of functional T cell plasticity and Th2 commitment in which we aimed to identify genes determining critical T cell fate decisions, potentially linking gene (dys)function to either autoimmune/inflammatory disease or functional persistence of cytotoxic T cells for cancer immunotherapy. Overall, the advanced CROP-Seq platform combining large-scale perturbations and targeted sequencing (TA) read-outs will catalyse a paradigm shift for CROP-Seq enabling high-throughput functional genomic screens that support the validation of drug targets in autoimmunity, inflammation and immuno-oncology. Citation Format: Nicole Untermoser, Johanna Irnstorfer, Anatoly Vasilyev, Nikola Vinko, Sumit Pawar, Anke Loregger, Adam Krejci, Henrik Schmidt, Tilmann Bürckstümmer. Single-cell CRISPR screens at scale to understand T cell biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB289.

PINK1 deficiency alters muscle stem cell fate decision and muscle regenerative capacity

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions.

Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

Intron detention tightly regulates the stemness/differentiation switch in the adult neurogenic niche

The adult mammalian brain retains some capacity to replenish neurons and glia, holding promise for brain regeneration. Thus, understanding the mechanisms controlling adult neural stem cell (NSC) differentiation is crucial. Paradoxically, adult NSCs in the subependymal zone transcribe genes associated with both multipotency maintenance and neural differentiation, but the mechanism that prevents conflicts in fate decisions due to these opposing transcriptional programmes is unknown. Here we describe intron detention as such control mechanism. In NSCs, while multiple mRNAs from stemness genes are spliced and exported to the cytoplasm, transcripts from differentiation genes remain unspliced and detained in the nucleus, and the opposite is true under neural differentiation conditions. We also show that m6A methylation is the mechanism that releases intron detention and triggers nuclear export, enabling rapid and synchronized responses. m6A RNA methylation operates as an on/off switch for transcripts with antagonistic functions, tightly controlling the timing of NSCs commitment to differentiation.

Gene-expression memory-based prediction of cell lineages from scRNA-seq datasets

Assigning single cell transcriptomes to cellular lineage trees by lineage tracing has transformed our understanding of differentiation during development, regeneration, and disease. However, lineage tracing is technically demanding, often restricted in time-resolution, and most scRNA-seq datasets are devoid of lineage information. Here we introduce Gene Expression Memory-based Lineage Inference (GEMLI), a computational tool allowing to robustly identify small to medium-sized cell lineages solely from scRNA-seq datasets. GEMLI allows to study heritable gene expression, to discriminate symmetric and asymmetric cell fate decisions and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. In human breast cancer biopsies, GEMLI reveals previously unknown gene expression changes at the onset of cancer invasiveness. The universal applicability of GEMLI allows studying the role of small cell lineages in a wide range of physiological and pathological contexts, notably in vivo. GEMLI is available as an R package on GitHub (https://github.com/UPSUTER/GEMLI).