Naive pluripotent stem cells (nPSCs) frequently undergo pathological loss of DNA methylation at imprinted gene loci, posing a hurdle for biomedical applications and underscoring the need to identify underlying causes. We show that nPSCs from inbred mouse strains exhibit strain-specific susceptibility to locus-specific deregulation of imprinting marks during reprogramming and upon exposure to a mitogen-activated protein kinase (MAPK) inhibitor, a common approach to maintain naive pluripotency. Analysis of genetically diverse nPSCs from the Diversity Outbred (DO) stock confirms the impact of genetic variation on epigenome stability, which we leverage to identify trans-acting quantitative trait loci (QTLs) that modulate DNA methylation levels at specific targets or genome-wide. Analysis of multi-target QTLs on chromosomes 4 and 17 suggests candidate transcriptional regulators contributing to DNA methylation maintenance in nPSCs. We propose that genetic variants represent biomarkers to identify pluripotent cell lines with desirable properties and may allow the targeted engineering of nPSCs with stable epigenomes.

Lung endothelial cells (ECs) and pericytes are closely juxtaposed with the respiratory epithelium before birth and thus may have instructive roles during development. To test this hypothesis, we screened EC-secreted proteins for their ability to alter cell differentiation in alveolar organoids. We identified secreted protein acidic and rich in cysteine-like protein 1 (SPARCL1) as an extracellular matrix molecule that can promote alveolar type 2 (AT2) cell differentiation in vitro. SPARCL1-treated organoids display lysozyme upregulation and a doubling in the number of AT2 cells at the expense of intermediate progenitors. SPARCL1 also induces the upregulation of nuclear factor κB (NF-κB) target genes, and suppression of NF-κB activation in lung organoids blocked SPARCL1 effects. NF-κB activation by lipopolysaccharide (LPS) was sufficient to induce AT2 cell differentiation; however, pharmacological inhibition of the pathway alone did not prevent it. These data support a role for SPARCL1 and NF-κB in alveolar cell differentiation and suggest a potential value in targeting this signaling axis to promote alveolar maturation and regeneration.

Spermatogenesis is driven by dramatic changes in chromatin regulation, gene transcription, and protein expression. To assess the mechanistic bases for these developmental changes, we utilized multiomic single-cell/nucleus RNA sequencing (sc/snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to identify chromatin changes associated with transcription in adult mouse and rat testes. We characterized the relationships between the transcriptomes and chromatin of both species, including the divergent expression of Id4 in spermatogonial stem cells between species. Promoter accessibility and gene expression showed the greatest association during meiosis in both species. We mapped the cross-species conservation of putative regulatory regions for key spermatogenic genes, including Cd9 and Spam1, and investigated correlations and disconnects in chromatin accessibility, gene expression, and protein expression via antibody-derived tags. Using a gene regulatory network (GRN) model, we identified 40 core regulons conserved between mouse and rat germ cells, highlighting the relevance of chromatin-related factors in regulating the transcription of canonical genes across spermatogenesis.

Neural stem cells (NSCs) of the subventricular zone (SVZ) could be a potential source for brain repair. These are heterogeneous cells with distinct activation states. To identify NSCs in the SVZ, different markers are used, including Gfap, Nestin, and Sox2. A comparison of these different methods to assess if the NSC marker used is selective toward specific NSC states is currently lacking. Here, we integrated six previously published single-cell RNA sequencing datasets from the adult mouse SVZ, where different methods were used to identify NSCs. Our data show that the approach used to isolate NSCs favors certain cell states over others. Our analyses underscore the importance of enriching for the NSC population of interest to increase data granularity. We also observed that cells with lower gene expression can be assigned incorrectly to clusters. We provide a framework for choosing the most optimal approach to enrich for NSC states of interest.

Duchenne muscular dystrophy (DMD) is a progressive myodegenerative disease that leads to severe muscle weakness and premature death. Mouse cardiosphere-derived cells (mCDCs) and extracellular vesicles (EVs) secreted by human cardiosphere-deriveds (hCDC-EVs) are therapeutic to mice with advanced-stage DMD. Here, we investigated the long-term benefits of monthly dosing when initiated early. At the endpoint, exercise performance and skeletal muscle function were strikingly preserved in mdx mice that had received mCDCs, but not in vehicle control. In contrast, the beneficial effects of hCDC-EVs waned after 6 months, in parallel with the development of anti-hCDC-EV antibodies. Further investigation showed that mCDCs lowered fibrosis and initiated a myogenic response program in mdx skeletal muscle. Thus, early and sustained intervention with mCDCs prevents disease progression for up to 1 year in mdx mice. This discovery offers new insights into how cell therapy can be used to treat DMD and motivates clinical testing of CDCs beginning in newly diagnosed DMD.

Advancements in genomics have revealed hundreds of loci associated with cardiovascular diseases, highlighting the role genetic variants play in disease pathogenesis. Notably, most variants lie within noncoding genomic regions that modulate transcription factor binding, chromatin accessibility, and thereby the expression levels and cell type specificity of gene transcripts. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful tool to delineate the pathogenicity of such variants and elucidate the underlying transcriptional mechanisms. Our review discusses the basics of noncoding variant-mediated pathogenesis, the methodologies utilized, and how hiPSC-based heart models can be leveraged to dissect the mechanisms of noncoding variants.

Nuclear reprogramming can change cellular fates. Yet, reprogramming efficiency is low, and the resulting cell types are often not functional. Here, we used nuclear transfer to eggs to follow single cells during reprogramming in vivo. We show that the differentiation success of reprogrammed cells varies across cell types and depends on the expression of genes specific to the previous cellular identity. We find subsets of reprogramming-resistant cells that fail to form functional cell types, undergo cell death, or disrupt normal body patterning. Reducing expression levels of genes specific to the cell type of origin leads to better reprogramming and improved differentiation trajectories. Thus, our work demonstrates that failing to reprogram in vivo is cell type specific and emphasizes the necessity of minimizing aberrant transcripts of the previous somatic identity for improving reprogramming.

Naive human pluripotent stem cells (hPSCs) model the pre-implantation epiblast. However, parent-specific epigenetic marks (imprints) are eroded in naive hPSCs, which represents an important deviation from the epiblast in vivo. To track the dynamics of imprint erasure during naive resetting in real time, we established a dual-colored fluorescent reporter at both alleles of the imprinted SNRPN locus. During primed-to-naive resetting, SNRPN expression becomes biallelic in most naive cells, and biallelic SNRPN expression is irreversible upon re-priming. We utilized this live-cell reporter to evaluate chemical and genetic strategies to minimize imprint erasure. Decreasing the level of MEK/ERK inhibition or overexpressing the KRAB zinc-finger protein ZFP57 protected a subset of imprints during naive resetting. Combining these two strategies protected imprint levels to a further extent than either strategy alone. This study offers an experimental tool to track and enhance imprint stability during transitions between human pluripotent states in vitro.

Here, we developed a CRISPR-Cas9 arrayed screen to investigate lipid handling pathways in human induced pluripotent stem cell (iPSC)-derived microglia. We established a robust method for the nucleofection of CRISPR-Cas9 ribonucleoprotein complexes into iPSC-derived myeloid cells, enabling genetic perturbations. Using this approach, we performed a targeted screen to identify key regulators of lipid droplet formation dependent on Apolipoprotein E (APOE). We identify the Mammalian Target of Rapamycin Complex 1 (mTORC1) signaling pathway as a critical modulator of lipid storage in both APOE3 and APOE knockout microglia. This study is a proof of concept underscoring the utility of CRISPR-Cas9 technology in elucidating the molecular pathways of lipid dysregulation associated with Alzheimer's disease and neuroinflammation.

Postnatal neocortical development is a complex period wherein radial glial progenitors (RGPs) complete excitatory neurogenesis and transition to the production of glia. Here, we take advantage of a multi-layered lineage tracing tool pbacBarcode, to examine the contributions of individual cortical RGPs to the postnatal cortex. We reveal that some individual cortical RGPs are multipotent and give rise to olfactory bulb interneurons, astrocytes, and oligodendrocytes in a ∼2:1:1 ratio. We provide evidence that differentiation potential into terminal cell types is maintained as late as post-natal day (P)4, suggesting that a population decline model, as opposed to cell fate restriction, underlies postnatal neocortical development. Moreover, a pool of proliferative intermediary cells, which may represent a multipotent postnatal intermediate progenitor cell population, may contribute to the production of the three major cell types. Lastly, we examine RGP postnatal contribution to oligodendrocytes and show that oligodendrocyte progenitor founder cell production by cortical RGPs is largely complete by P3.

Myelin transcription factor 1 like (MYT1L) is a neuronal transcription factor highly expressed in the developing and adult brain, and, while pathogenic MYT1L mutations cause neurodevelopmental disorders, these have not been characterized in human models of neurodevelopment. Here, we modeled the consequences of pathogenic MYT1L mutation using human stem cell-derived cortical neurons, demonstrating that MYT1L mutation alters the differentiation trajectory, increasing neuronal gene expression, morphological complexity, and synapse production. We also examined consequences of MYT1L mutation in mature cortical interneurons, identifying hallmarks of impaired neuronal identity and maturation and correspondingly altered channel expression and electrophysiological properties. Finally, by defining MYT1L genome-wide occupancy in cortical interneurons, we identified direct MYT1L targets likely to mediate these phenotypes. Together, this work elucidates new MYT1L requirements for human cortical interneuron development and demonstrates how pathogenic MYT1L mutation perturbs this developmental program, contributing to the etiology of neurodevelopmental disorders.

Radiation induces clonal hematopoiesis (CH) involving high-frequency somatic mutations in hematopoietic cells. However, the effects of radiation on clonal expansion of hematopoietic progenitor cells and lymphocytes remain elusive. Here, we investigate CH mutations and T cell receptor (TCR) and B cell receptor (BCR) sequences within the bone marrow cells of mice 18 months after irradiation (3 Gy) and age-matched controls. Two to six CH mutations were identified in the irradiated mice (N = 5), while only one of the four control mice carried a CH mutation. These CH mutations detected in the bone marrow were also identified in the splenic CD11b myeloid cell population. Meanwhile, the cumulative size of the ten largest TCR and BCR clones, as well as their clonality, did not differ significantly between irradiated and control mice. Our findings suggest that radiation preferentially induces clonal expansion of hematopoietic progenitor cells over mature lymphocytes in the bone marrow.

Stem cell-based models of human heart tissue and cardiac differentiation employ monolayer and 3D organoid cultures with different properties, cell type composition, and maturity. Here we show how cardiac monolayer, embryoid body, and engineered heart tissue trajectories compare in a single-cell roadmap of atrial and ventricular differentiation conditions. Using a multiomic approach and gene-regulatory network inference, we identified regulators of the epicardial, atrial, and ventricular cardiomyocyte lineages. We identified ZNF711 as a regulatory switch and safeguard for cardiomyocyte commitment. We show that ZNF711 ablation prevents cardiomyocyte differentiation in the absence of retinoic acid, causing progenitors to be diverted more prominently to epicardial and other lineages. Retinoic acid rescues this shift in lineage commitment and promotes atrial cardiomyocyte differentiation by regulation of shared and complementary target genes, showing interplay between ZNF711 and retinoic acid in cardiac lineage commitment.

Subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells has demonstrated therapeutic potential in macular degeneration. However, its efficiency is limited in wet age-related macular degeneration (wet AMD) due to choroidal neovascularization (CNV). To investigate the feasibility of hESC-RPE cell transplantation, we employed a surgical approach to induce retinal detachment, which allowed the removal of CNV lesions. After retinal reattachment, hESC-RPE cells were transplanted into the subretinal space. Ten patients were enrolled and divided into 2 groups. No retinal edema or CNV recurrence was observed in group 1 (7 patients without bleeding). Group 2 (3 patients with bleeding) had persistent fundus inflammation, and one patient experienced CNV recurrence. All patients were managed effectively without vision loss. These findings suggest that subretinal transplantation of hESC-RPE cells after CNV removal is safe and well tolerated; however, damage caused during CNV removal may trigger persistent inflammation and CNV recurrence. This study was registered at ClinicalTrials.gov (NCT02749734).

Photoreceptor degeneration is a leading cause of untreatable sight loss. Previously, we showed that human pluripotent stem cell-derived cone photoreceptors (hCones) can rescue retinal function in the Rd1 mouse model of rod-cone dystrophy. However, retinal degenerations display markedly different severities and concomitant remodeling of the remaining retina; for photoreceptor replacement therapy to be broadly effective, it must work for a variety of disease phenotypes. Here, we sought to rescue the Aipl1 model of Leber congenital amaurosis, a particularly fast, severe condition. After transplantation of hCones, host cone bipolar cells underwent extensive remodeling and formed nascent synaptic-like connections. Electrophysiological recordings showed robust rescue of light-evoked activity across visually relevant photopic intensities, and treated mice exhibited visually evoked optokinetic head-tracking behavior. Thus, human cone photoreceptor replacement therapy is feasible even in very severe cases of retinal dystrophy, offering promise as a disease-agnostic therapy in Leber congenital amaurosis (LCA) and in other advanced retinal degenerations.

Proper development of surface epithelium (SE) is a requisite for the normal development and function of ectodermal appendages; however, the molecular mechanisms underlying SE commitment remain largely unexplored. Here, we developed a KRT8 reporter system and utilized it to identify FOXO4 and SP6 as novel, essential regulators governing SE commitment. We found that the FOXO4-SP6 axis governs SE fate and its abrogation markedly impedes SE fate determination. Mechanistically, FOXO4 regulates SE initiation by shaping the SE chromatin accessibility landscape and regulating the deposition of H3K4me3. SP6, as a novel effector of FOXO4, activates SE-specific genes through modulating the H3K27ac deposition across their super-enhancers. Our work highlights the regulatory function of the FOXO4-SP6 axis in SE development, contributing to an improved understanding of SE fate decisions and providing a research foundation for the therapeutic application of ectodermal dysplasia.

The extracellular matrix-F-actin-Yes-associated protein 1 (YAP1)-Notch mechanosignaling axis is a gatekeeper in the fate decisions of bipotent pancreatic progenitors (bi-PPs). However, the link between F-actin dynamics and YAP1 activity remains poorly understood. Here, we identify salt-inducible kinases (SIKs) as mediators of F-actin-triggered changes in YAP1 activity. Interestingly, sodium chloride treatment promotes the differentiation of bi-PPs into NEUROG3 endocrine progenitors (EPs) through enhanced SIK expression. Consistently, the pan-SIK inhibitor HG-9-09-01 (HG) inhibits latrunculin B (LatB)-induced EP differentiation via nuclear YAP1 accumulation. Unexpectedly, withdrawal of HG after a 12-h treatment increased SIK expression by a negative feedback mechanism, leading to significantly enhanced endocrinogenesis. Therefore, the combined treatment of bi-PPs with LatB and HG for 12 h boosted endocrinogenesis, ultimately leading to an increased number of beta cells. In summary, we identify SIKs as new transducers of mechanotransduction-triggered induction of pancreatic endocrine cell fates.

Outer retinal degenerative diseases (RDDs) and injuries leading to photoreceptor (PR) loss are prevailing causes of blindness worldwide. While significant progress has been made in the manufacture of human pluripotent stem cell (hPSC)-derived PRs, robust production of pluripotent stem cell (PSC)-PRs from swine, a popular preclinical large animal model, would provide an avenue to collect conspecific functional and safety data to complement human xenograft studies. Toward this goal, we describe the highly efficient generation of PR-dominant porcine induced PSC (piPSC)-derived retinal organoids (ROs) using modifications of our established hPSC-RO differentiation protocol. Porcine iPSC-ROs were characterized using immunocytochemistry (ICC) and single-cell RNA sequencing (scRNA-seq), which revealed the presence and maturation of major neural retina cell types, including PRs and retinal ganglion cells, which possess molecular signatures akin to those found in hPSC-ROs. In late piPSC-ROs, a highly organized outer neuroepithelium was observed with rods and cones possessing outer segments and axon terminals expressing pre-synaptic markers adjacent to dendritic terminals of bipolar cells. The existence of piPSC lines and protocols that support reproducible, scalable production of female and male ROs will facilitate transplant studies in porcine models of retinal injury and RDDs unconfounded by immunological and evolutionary incompatibilities inherent to human xenografts.

Mutations in mitochondrial DNA cause severe multisystem disease frequently associated with muscle weakness. The m.3243A>G mutation is the major cause of mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). Experimental models that recapitulate the disease phenotype in vitro for disease modeling or drug screening are very limited. We have therefore generated hiPSC-derived muscle fibers with variable heteroplasmic mtDNA mutation load without significantly affecting muscle differentiation potential. The cells exhibit physiological characteristics of muscle fibers and show a well-organized myofibrillar structure. In cells carrying the m.3243A>G mutation, the mitochondrial membrane potential and oxygen consumption were reduced in relation to the mutant load. We have shown through proteomic, phosphoproteomic, and metabolomic analyses that the m.3243A>G mutation variably affects the cell phenotype in relation to the mutant load. This variation is reflected by an increase in the NADH/NAD ratio, which in turn influences key nutrient-sensing pathways in the myofibers. This model enables a detailed study of the impact of the mutation on cellular bioenergetics and on muscle physiology with the potential to provide a platform for drug screening.