Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells.

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

Dark force rising: Reawakening and targeting of fetal-like stem cells in colorectal cancer.

Stem cells play pivotal roles in maintaining intestinal homeostasis, orchestrating regeneration, and in key steps of colorectal cancer (CRC) initiation and progression. Intriguingly, adult stem cells are reduced during many of these processes. On the contrary, primitive fetal programs, commonly detected in development, emerge during tissue repair, CRC metastasis, and therapy resistance. Recent findings indicate a dynamic continuum between adult and fetal stem cell programs. We discuss critical mechanisms facilitating the plasticity between stem cell states and highlight the heterogeneity observed upon the appearance of fetal-like states. We focus on therapeutic opportunities that arise by targeting fetal-like CRC cells and how those concepts can be translated into the clinic.

Kmt2c restricts G-CSF-driven HSC mobilization and granulocyte production in a methyltransferase-independent manner.

Granulocyte colony-stimulating factor (G-CSF) is widely used to enhance myeloid recovery after chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Unfortunately, through the course of chemotherapy, cancer patients can acquire leukemogenic mutations that cause therapy-related myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). This raises the question of whether therapeutic G-CSF might potentiate therapy-related MDS/AML by disproportionately stimulating mutant HSCs and other myeloid progenitors. A common mutation in therapy-related MDS/AML involves chromosome 7 deletions that inactivate many tumor suppressor genes, including KMT2C. Here, we show that Kmt2c deletions hypersensitize murine HSCs and myeloid progenitors to G-CSF, as evidenced by increased HSC mobilization and enhanced granulocyte production from granulocyte-monocyte progenitors (GMPs). Furthermore, Kmt2c attenuates the G-CSF response independently from its SET methyltransferase function. Altogether, the data raise concerns that monosomy 7 can hypersensitize progenitors to G-CSF, such that clinical use of G-CSF may amplify the risk of therapy-related MDS/AML.

LEUTX regulates porcine embryonic genome activation in somatic cell nuclear transfer embryos.

Emerging evidence highlights the regulatory role of paired-like (PRD-like) homeobox transcription factors (TFs) in embryonic genome activation (EGA). However, the majority of PRD-like genes are lost in rodents, thus prompting an investigation into PRD-like TFs in other mammals. Here, we showed that PRD-like TFs were transiently expressed during EGA in human, monkey, and porcine fertilized embryos, yet they exhibited inadequate expression in their cloned embryos. This study, using pig as the research model, identified LEUTX as a key PRD-like activator of porcine EGA through genomic profiling and found that LEUTX overexpression restored EGA failure and improved preimplantation development and cloning efficiency in porcine cloned embryos. Mechanistically, LEUTX opened EGA-related genomic regions and established histone acetylation via recruiting acetyltransferases p300 and KAT2A. These findings reveal the regulatory mechanism of LEUTX to govern EGA in pigs, which may provide valuable insights into the study of early embryo development for other non-rodent mammals.

hnRNPU is required for spermatogonial stem cell pool establishment in mice.

The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.

The dynamic landscape of enhancer-derived RNA during mouse early embryo development.

Enhancer-derived RNAs (eRNAs) play critical roles in diverse biological processes by facilitating their target gene expression. However, the abundance and function of eRNAs in early embryos are not clear. Here, we present a comprehensive eRNA atlas by systematically integrating publicly available datasets of mouse early embryos. We characterize the transcriptional and regulatory network of eRNAs and show that different embryo developmental stages have distinct eRNA expression and regulatory profiles. Paternal eRNAs are activated asymmetrically during zygotic genome activation (ZGA). Moreover, we identify an eRNA, MZGAe1, which plays an important function in regulating mouse ZGA and early embryo development. MZGAe1 knockdown leads to a developmental block from 2-cell embryo to blastocyst. We create an online data portal, M2ED2, to query and visualize eRNA expression and regulation. Our study thus provides a systematic landscape of eRNA and reveals the important role of eRNAs in regulating mouse early embryo development.

Unraveling hallmark suitability for staging pre- and post-implantation stem cell models.

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.

IL-4-induced SOX9 confers lineage plasticity to aged adult lung stem cells.

Wound healing in response to acute injury is mediated by the coordinated and transient activation of parenchymal, stromal, and immune cells that resolves to homeostasis. Environmental, genetic, and epigenetic factors associated with inflammation and aging can lead to persistent activation of the microenvironment and fibrosis. Here, we identify opposing roles of interleukin-4 (IL-4) cytokine signaling in interstitial macrophages and type II alveolar epithelial cells (ATIIs). We show that IL4Ra signaling in macrophages promotes regeneration of the alveolar epithelium after bleomycin-induced lung injury. Using organoids and mouse models, we show that IL-4 directly acts on a subset of ATIIs to induce the expression of the transcription factor SOX9 and reprograms them toward a progenitor-like state with both airway and alveolar lineage potential. In the contexts of aging and bleomycin-induced lung injury, this leads to aberrant epithelial cell differentiation and bronchiolization, consistent with cellular and histological changes observed in interstitial lung disease.

Parasitoid cues modulate Drosophila germline development and stem cell proliferation.

Environmental factors influence an organism's reproductive ability by regulating germline development and physiology. While the reproductive adaptations in response to extrinsic stress cues offer fitness and survival advantages to individuals, the mechanistic understanding of these modifications remains unclear. Here, we find that parasitoid wasps' stress signaling regulates Drosophila melanogaster oogenesis. We show that fruit flies dwelling in the wasp-infested area elevate their fecundity, and the observed reproductive response is specific to Pachycrepoideus sp., a pupal parasitoid wasp. Pachycrepoideus-specific olfactory and visual cues recruit the signaling pathways that promote germline stem cell proliferation and accelerate follicle development, increasing egg production in Drosophila females. Downregulation of signaling engaged in oocyte development by shifting flies to a non-wasp-infested environment increases apoptosis of the developing follicles. Thus, this study establishes host germline responsiveness to parasitoid-specific signals and supports a predator strategy to increase hosts for infection.

ptx3a+ fibroblast/epicardial cells provide a transient macrophage niche to promote heart regeneration.

Macrophages conduct critical roles in heart repair, but the niche required to nurture and anchor them is poorly studied. Here, we investigated the macrophage niche in the regenerating heart. We analyzed cell-cell interactions through published single-cell RNA sequencing datasets and identified a strong interaction between fibroblast/epicardial (Fb/Epi) cells and macrophages. We further visualized the association of macrophages with Fb/Epi cells and the blockage of macrophage response without Fb/Epi cells in the regenerating zebrafish heart. Moreover, we found that ptx3a+ epicardial cells associate with reparative macrophages, and their depletion resulted in fewer reparative macrophages. Further, we identified csf1a expression in ptx3a+ cells and determined that pharmacological inhibition of the csf1a pathway or csf1a knockout blocked the reparative macrophage response. Moreover, we found that genetic overexpression of csf1a enhanced the reparative macrophage response with or without heart injury. Altogether, our studies illuminate a cardiac Fb/Epi niche, which mediates a beneficial macrophage response after heart injury.

A transcription factor atlas of stem cell fate in planarians.

Whole-body regeneration requires the ability to produce the full repertoire of adult cell types. The planarian Schmidtea mediterranea contains over 125 cell types, which can be regenerated from a stem cell population called neoblasts. Neoblast fate choice can be regulated by the expression of fate-specific transcription factors (FSTFs). How fate choices are made and distributed across neoblasts versus their post-mitotic progeny remains unclear. We used single-cell RNA sequencing to systematically map fate choices made in S/G2/M neoblasts and, separately, in their post-mitotic progeny that serve as progenitors for all adult cell types. We defined transcription factor expression signatures associated with all detected fates, identifying numerous new progenitor classes and FSTFs that regulate them. Our work generates an atlas of stem cell fates with associated transcription factor signatures for most cell types in a complete adult organism.

Adipose stem cells control obesity-induced T cell infiltration into adipose tissue.

T cell infiltration into white adipose tissue (WAT) drives obesity-induced adipose inflammation, but the mechanisms of obesity-induced T cell infiltration into WAT remain unclear. Our single-cell RNA sequencing reveals a significant impact of adipose stem cells (ASCs) on T cells. Transplanting ASCs from obese mice into WAT enhances T cell accumulation. C-C motif chemokine ligand 5 (CCL5) is upregulated in ASCs as early as 4 weeks of high-fat diet feeding, coinciding with the onset of T cell infiltration into WAT during obesity. ASCs and bone marrow transplantation experiments demonstrate that CCL5 from ASCs plays a crucial role in T cell accumulation during obesity. The production of CCL5 in ASCs is induced by tumor necrosis factor alpha via the nuclear factor κB pathway. Overall, our findings underscore the pivotal role of ASCs in regulating T cell accumulation in WAT during the early phases of obesity, emphasizing their importance in modulating adaptive immunity in obesity-induced adipose inflammation.

Regulation of muscle hypertrophy through granulin: Relayed communication among mesenchymal progenitors, macrophages, and satellite cells.

Skeletal muscles exert remarkable regenerative or adaptive capacities in response to injuries or mechanical loads. However, the cellular networks underlying muscle adaptation are poorly understood compared to those underlying muscle regeneration. We employed single-cell RNA sequencing to investigate the gene expression patterns and cellular networks activated in overloaded muscles and compared these results with those observed in regenerating muscles. The cellular composition of the 4-day overloaded muscle, when macrophage infiltration peaked, closely resembled that of the 10-day regenerating muscle. In addition to the mesenchymal progenitor-muscle satellite cell (MuSC) axis, interactome analyses or targeted depletion experiments revealed communications between mesenchymal progenitors-macrophages and macrophages-MuSCs. Furthermore, granulin, a macrophage-derived factor, inhibited MuSC differentiation, and Granulin-knockout mice exhibited blunted muscle hypertrophy due to the premature differentiation of overloaded MuSCs. These findings reveal the critical role of granulin through the relayed communications of mesenchymal progenitors, macrophages, and MuSCs in facilitating efficient muscle hypertrophy.

ABHD7-mediated depalmitoylation of lamin A promotes myoblast differentiation.

LMNA gene mutation can cause muscular dystrophy, and post-translational modification plays a critical role in regulating its function. Here, we identify that lamin A is palmitoylated at cysteine 522, 588, and 591 residues, which are reversely catalyzed by palmitoyltransferase zinc finger DHHC-type palmitoyltransferase 5 (ZDHHC5) and depalmitoylase α/ß hydrolase domain 7 (ABHD7). Furthermore, the metabolite lactate promotes palmitoylation of lamin A by inhibiting the interaction between it and ABHD7. Interestingly, low-level palmitoylation of lamin A promotes, whereas high-level palmitoylation of lamin A inhibits, murine myoblast differentiation. Together, these observations suggest that ABHD7-mediated depalmitoylation of lamin A controls myoblast differentiation.

Trans-omic profiling uncovers molecular controls of early human cerebral organoid formation.

Defining the molecular networks orchestrating human brain formation is crucial for understanding neurodevelopment and neurological disorders. Challenges in acquiring early brain tissue have incentivized the use of three-dimensional human pluripotent stem cell (hPSC)-derived neural organoids to recapitulate neurodevelopment. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the exit of pluripotency and neural differentiation toward human cerebral organoids (hCOs). These data reveal key phospho-signaling events and their convergence on transcriptional factors to regulate hCO formation. Comparative analysis with developing human and mouse embryos demonstrates the fidelity of our hCOs in modeling embryonic brain development. Finally, we demonstrate that biochemical modulation of AKT signaling can control hCO differentiation. Together, our data provide a comprehensive resource to study molecular controls in human embryonic brain development and provide a guide for the future development of hCO differentiation protocols.

G2 arrest primes hematopoietic stem cells for megakaryopoiesis.

In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.

Alternative mRNA splicing events and regulators in epidermal differentiation.

Alternative splicing (AS) of messenger RNAs occurs in ∼95% of multi-exon human genes and generates diverse RNA and protein isoforms. We investigated AS events associated with human epidermal differentiation, a process crucial for skin function. We identified 6,413 AS events, primarily involving cassette exons. We also predicted 34 RNA-binding proteins (RBPs) regulating epidermal AS, including 19 previously undescribed candidate regulators. From these results, we identified FUS as an RBP that regulates the balance between keratinocyte proliferation and differentiation. Additionally, we characterized the function of a cassette exon AS event in MAP3K7, which encodes a kinase involved in cell signaling. We found that a switch from the short to long isoform of MAP3K7, triggered during differentiation, enforces the demarcation between proliferating basal progenitors and overlying differentiated strata. Our findings indicate that AS occurs extensively in the human epidermis and has critical roles in skin homeostasis.

Baf155 controls hematopoietic differentiation and regeneration through chromatin priming.

Chromatin priming promotes cell-type-specific gene expression, lineage differentiation, and development. The mechanism of chromatin priming has not been fully understood. Here, we report that mouse hematopoietic stem and progenitor cells (HSPCs) lacking the Baf155 subunit of the BAF (BRG1/BRM-associated factor) chromatin remodeling complex produce a significantly reduced number of mature blood cells, leading to a failure of hematopoietic regeneration upon transplantation and 5-fluorouracil (5-FU) injury. Baf155-deficient HSPCs generate particularly fewer neutrophils, B cells, and CD8+ T cells at homeostasis, supporting a more immune-suppressive tumor microenvironment and enhanced tumor growth. Single-nucleus multiomics analysis reveals that Baf155-deficient HSPCs fail to establish accessible chromatin in selected regions that are enriched for putative enhancers and binding motifs of hematopoietic lineage transcription factors. Our study provides a fundamental mechanistic understanding of the role of Baf155 in hematopoietic lineage chromatin priming and the functional consequences of Baf155 deficiency in regeneration and tumor immunity.