Photo-Crosslinked Pro-Angiogenic Hydrogel Dressing for Wound Healing.

Severe burns are one of the most devastating injuries, in which sustained inflammation and ischemia often delay the healing process. Pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) have been widely studied for promoting wound healing. However, the short half-life and instability of VEGF limit its clinical applications. In this study, we develop a photo-crosslinked hydrogel wound dressing from methacrylate hyaluronic acid (MeHA) bonded with a pro-angiogenic prominin-1-binding peptide (PR1P). The materials were extruded in wound bed and in situ formed a wound dressing via exposure to short-time ultraviolet radiation. The study shows that the PR1P-bonded hydrogel significantly improves VEGF recruitment, tubular formation, and cell migration in vitro. Swelling, Scanning Electron Microscope, and mechanical tests indicate the peptide does not affect the overall mechanical and physical properties of the hydrogels. For in vivo studies, the PR1P-bonded hydrogel dressing enhances neovascularization and accelerates wound closure in both deep second-degree burn and full-thickness excisional wound models. The Western blot assay shows such benefits can be related to the activation of the VEGF-Akt signaling pathway. These results suggest this photo-crosslinked hydrogel dressing efficiently promotes VEGF recruitment and angiogenesis in skin regeneration, indicating its potential for clinical applications in wound healing.

Simultaneous profiling of RNA isoforms and chromatin accessibility of single cells of human retinal organoids.

Single-cell multi-omics sequencing is a powerful approach to analyze complex mechanisms underlying neuronal development and regeneration. However, current methods lack the ability to simultaneously profile RNA alternative splicing and chromatin accessibility at the single-cell level. We develop a technique, single-cell RNA isoform and chromatin accessibility sequencing (scRICA-seq), which demonstrates higher sensitivity and cost-effectiveness compared to existing methods. scRICA-seq can profile both isoforms and chromatin accessibility for up to 10,000 single cells in a single run. Applying this method to human retinal organoids, we construct a multi-omic cell atlas and reveal associations between chromatin accessibility, isoform expression of fate-determining factors, and alternative splicing events in their binding sites. This study provides insights into integrating epigenetics, transcription, and RNA splicing to elucidate the mechanisms underlying retinal neuronal development and fate determination.

Effects of ECM protein-coated surfaces on the generation of retinal pigment epithelium cells differentiated from human pluripotent stem cells.

Retinal degeneration diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), initially manifest as dysfunction or death of the retinal pigment epithelium (RPE). Subretinal transplantation of human pluripotent stem cell (hPSC)-derived RPE cells has emerged as a potential therapy for retinal degeneration. However, RPE cells differentiated from hPSCs using current protocols are xeno-containing and are rarely applied in clinical trials. The development of hPSC-derived RPE cell differentiation protocols using xeno-free biomaterials is urgently needed for clinical applications. In this study, two protocols (the activin A and NIC84 protocols) were selected for modification and use in the differentiation of hiPSCs into RPE cells; the chetomin concentration was gradually increased to achieve high differentiation efficiency of RPE cells. The xeno-free extracellular matrix (ECM) proteins, laminin-511, laminin-521 and recombinant vitronectin, were selected as plate-coating substrates, and a Matrigel (xeno-containing ECM)-coated surface was used as a positive control. Healthy, mature hPSC-derived RPE cells were transplanted into 21-day-old Royal College of Surgeons (RCS) rats, a model of retinal degeneration disease. The visual function of RCS rats was evaluated by optomotor response (qOMR) and electroretinography after transplantation of hPSC-derived RPE cells. Our study demonstrated that hPSCs can be efficiently differentiated into RPE cells on LN521-coated dishes using the NIC84 protocol, and that subretinal transplantation of the cell suspensions can delay the progression of vision loss in RCS rats.

Integrating Complaint Analysis into Hospital Management: A Comparative Study of Surgical and Non-Surgical Complaints.

Aims/Background: In an era where patient-centred care is paramount, effectively managing and analyzing hospital complaints is crucial for improving service quality and patient satisfaction. This study examines hospital complaints to enhance management practices by differentiating between surgery-related and non-surgery-related grievances. By identifying patterns in complaint types and outcomes, we aim to inform targeted quality improvement strategies that address specific patient concerns and boost operational efficiency. Methods: The study utilized data from an internal complaint management system over one year. Complaints were categorized as either surgery-related or non-surgery-related. Descriptive statistics and cross-tabulation analysis were employed to examine the data. The sample comprised 132 complaints, with 67 being surgery-related and 65 non-surgery-related. Results: The analysis revealed that surgery-related complaints frequently involved issues with 'Patient Communication' and 'Surgical Error', while non-surgery-related complaints were primarily about the 'Medical Treatment Process'. The Surgery Department received the highest number of complaints, indicating a critical area for intervention. Additionally, the correlation between complaint types and outcomes provided insights into potential areas for improvement. Conclusion: The findings highlight the need for targeted communication training and procedural enhancements in surgical departments. Non-surgical departments should focus on improving treatment protocols and transparency. These strategies can reduce complaints and improve patient satisfaction. Future research should develop and test interventions based on these insights to further enhance healthcare quality.

Comparison of retinal degeneration treatment with four types of different mesenchymal stem cells, human induced pluripotent stem cells and RPE cells in a rat retinal degeneration model.

BACKGROUND: Retinal degeneration (RD) is a group of disorders on irreversible vision loss. Multiple types of stem cells were used in clinical trials for RD treatment. However, it remains unknown what kinds of stem cells are most effective for the treatment. Therefore, we investigated the subretinal transplantation of several types of stem cells, human adipose-derived stem cells (hADSCs), amniotic fluid stem cells (hAFSCs), bone marrow stem cells (hBMSCs), dental pulp stem cells (hDPSCs), induced pluripotent stem cell (hiPSC), and hiPSC-derived retinal pigment epithelium (RPE) cells for protection effects, paracrine effects and treatment efficiency in an RD disease model rats. METHODS: The generation and characterization of these stem cells and hiPSC-derived RPE cells were performed before transplantation. The stem cells or hiPSC-derived RPE cell suspension labelled with CellTracker Green to detect transplanted cells were delivered into the subretinal space of 3-week-old RCS rats. The control group received subretinal PBS injection or non-injection. A series of detections including fundus photography, optomotor response (OMR) evaluations, light-dark box testing, electroretinography (ERG), and hematoxylin and eosin (HE) staining of retinal sections were conducted after subretinal injection of the cells. RESULTS: Each stem cell, hiPSC-derived RPE cell or PBS (blank experiment) was successfully transplanted into at least six RCS rats subretinally. Compared with the control rats, RCS rats subjected to subretinal transplantation of any stem cells except hiPSCs showed higher ERG waves (p < 0.05) and quantitative OMR (qOMR) index values (hADSCs: 1.166, hAFSCs: 1.249, hBMSCs: 1.098, hDPSCs: 1.238, hiPSCs: 1.208, hiPSC-RPE cells: 1.294, non-injection: 1.03, PBS: 1.06), which indicated better visual function, at 4 weeks post-injection. However, only rats that received hiPSC-derived RPE cells maintained their visual function at 8 weeks post-injection (p < 0.05). The outer nuclear layer thickness observed in histological sections after HE staining showed the same pattern as the ERG and qOMR results. CONCLUSIONS: Compared to hiPSC-derived RPE cells, adult and fetal stem cells yielded improvements in visual function for up to 4 weeks post-injection; this outcome was mainly based on the paracrine effects of several types of growth factors secreted by the stem cells. Patients with RD will benefit from the stem cell therapy.

Brain methylome remodeling selectively regulates neuronal activity genes linking to emotional behaviors in mice exposed to maternal immune activation.

How early life experience is translated into storable epigenetic information leading to behavioral changes remains poorly understood. Here we found that Zika virus (ZIKV) induced-maternal immune activation (MIA) imparts offspring with anxiety- and depression-like behavior. By integrating bulk and single-nucleus RNA sequencing (snRNA-seq) with genome-wide 5hmC (5-hydroxymethylcytosine) profiling and 5mC (5-methylcytosine) profiling in prefrontal cortex (PFC) of ZIKV-affected male offspring mice, we revealed an overall loss of 5hmC and an increase of 5mC levels in intragenic regions, associated with transcriptional changes in neuropsychiatric disorder-related genes. In contrast to their rapid initiation and inactivation in normal conditions, immediate-early genes (IEGs) remain a sustained upregulation with enriched expression in excitatory neurons, which is coupled with increased 5hmC and decreased 5mC levels of IEGs in ZIKV-affected male offspring. Thus, MIA induces maladaptive methylome remodeling in brain and selectively regulates neuronal activity gene methylation linking to emotional behavioral abnormalities in offspring.

Single-Cell RNA-Sequencing Provides Insight into Skeletal Muscle Evolution during the Selection of Muscle Characteristics.

Skeletal muscle comprises a large, heterogeneous assortment of cell populations that interact to maintain muscle homeostasis, but little is known about the mechanism that controls myogenic development in response to artificial selection. Different pig (Sus scrofa) breeds exhibit distinct muscle phenotypes resulting from domestication and selective breeding. Using unbiased single-cell transcriptomic sequencing analysis (scRNA-seq), the impact of artificial selection on cell profiles is investigated in neonatal skeletal muscle of pigs. This work provides panoramic muscle-resident cell profiles and identifies novel and breed-specific cells, mapping them on pseudotime trajectories. Artificial selection has elicited significant changes in muscle-resident cell profiles, while conserving signs of generational environmental challenges. These results suggest that fibro-adipogenic progenitors serve as a cellular interaction hub and that specific transcription factors identified here may serve as candidate target regulons for the pursuit of a specific muscle phenotype. Furthermore, a cross-species comparison of humans, mice, and pigs illustrates the conservation and divergence of mammalian muscle ontology. The findings of this study reveal shifts in cellular heterogeneity, novel cell subpopulations, and their interactions that may greatly facilitate the understanding of the mechanism underlying divergent muscle phenotypes arising from artificial selection.

Chemical-induced phase transition and global conformational reorganization of chromatin.

Chemicals or drugs can accumulate within biomolecular condensates formed through phase separation in cells. Here, we use super-resolution imaging to search for chemicals that induce phase transition within chromatin at the microscale. This microscopic screening approach reveals that adriamycin (doxorubicin) - a widely used anticancer drug that is known to interact with chromatin - specifically induces visible local condensation and global conformational change of chromatin in cancer and primary cells. Hi-C and ATAC-seq experiments systematically and quantitatively demonstrate that adriamycin-induced chromatin condensation is accompanied by weakened chromatin interaction within topologically associated domains, compartment A/B switching, lower chromatin accessibility, and corresponding transcriptomic changes. Mechanistically, adriamycin complexes with histone H1 and induces phase transition of H1, forming fibrous aggregates in vitro. These results reveal a phase separation-driven mechanism for a chemotherapeutic drug.

Culture conditions of mouse ESCs impact the tumor appearance in vivo.

Various culture conditions by small molecules have been explored to extend pluripotency of stem cells, but their impacts on cell fate in vivo remain elusive. We systematically compared the effects of various culture conditions on the pluripotency and cell fate in vivo of mouse embryonic stem cells (ESCs) by tetraploid embryo complementation assay. Conventional ESC cultures in serum/LIF-based condition produced complete ESC mice and also the survival to adulthood at the highest rates of all other chemical-based cultures. Moreover, long-term examination of the survived ESC mice demonstrated that conventional ESC cultures did not lead to visible abnormality for up to 1.5-2 years, whereas the prolonged chemical-based cultures developed retroperitoneal atypical teratomas or leiomyomas. The chemical-based cultures exhibited transcriptomes and epigenomes that typically differed from those of conventional ESC cultures. Our results warrant further refinement of culture conditions in promoting the pluripotency and safety of ESCs in future applications.

Distinct patterns of responses in endothelial cells and smooth muscle cells following vascular injury.

Vascular smooth muscle cells (SMCs) are heterogeneous, and their differential responses to vascular injury are not well understood. To address this question, we performed single-cell analysis of vascular cells to a ligation injury in mouse carotid arteries after 3 days. While endothelial cells had a homogeneous activation of mesenchymal genes, less than 30% of SMCs responded to the injury and generated 2 distinct clusters - i.e., proinflammatory SMCs and stress-responsive SMCs. Proinflammatory SMCs were enriched with high levels of inflammatory markers such as vascular cell adhesion molecule-1 while stress-responsive SMCs overexpressed heat shock proteins. Trajectory analysis suggested that proinflammatory SMCs were potentially derived from a specific subpopulation of SMCs. Ligand-receptor pair analysis showed that the interaction between macrophages and proinflammatory SMCs was the major cell-cell communication among all cell types in the injured arteries. In vitro coculture demonstrated that VCAM1+ SMCs had a stronger chemotactic effect on macrophage recruitment than VCAM1- SMCs. Consistently, the number of VCAM1+ SMCs significantly increased in injured arteries and atherosclerotic lesions of ApoE-/- mice and human arteries. These findings provide insights at the single-cell level on the distinct patterns of endothelial cells and SMC responses to vascular injury.

Global Transcriptional and Epigenetic Reconfiguration during Chemical Reprogramming of Human Retinal Pigment Epithelial Cells into Photoreceptor-like Cells.

Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising therapies. Multiple types of stem and somatic cells have been tested for photoreceptor conversion. However, current induction efficiencies are still low and the molecular mechanisms underlying reprogramming remain to be clarified. In this work, by combining treatment with small molecules, we directly reprogrammed human fetal retinal pigment epithelial (RPE) cells into chemically induced photoreceptor-like cells (CiPCs) in vitro. Bulk and single-cell RNA sequencing, as well as methylation sequencing, were performed to understand the transcriptional and epigenetic changes during CiPCs conversion. A multi-omics analysis showed that the direct reprogramming process partly resembled events of early retina development. We also found that the efficiency of CiPCs conversion from RPE is much better than that from human dermal fibroblasts (HDF). The small molecules effectively induced RPE cells into CiPCs via suppression of the epithelial-to-mesenchymal transition (EMT). Among the signaling pathways involved in CiPCs conversion, glutamate receptor activation is prominent. In summary, RPE cells can be efficiently reprogrammed into photoreceptor-like cells through defined pharmacological modulations, providing a useful cell source for photoreceptor generation in cell replacement therapy for retinal degenerative diseases.

Single Molecule RNA Localization and Translation in the Mammalian Oocyte and Embryo.

During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.

Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus.

Zika virus (ZIKV) infection during pregnancy is linked to various developmental brain disorders. Infants who are asymptomatic at birth might have postnatal neurocognitive complications. However, animal models recapitulating these neurocognitive phenotypes are lacking, and the circuit mechanism underlying behavioral abnormalities is unknown. Here, we show that ZIKV infection during mouse pregnancy induces maternal immune activation (MIA) and leads to autistic-like behaviors including repetitive self-grooming and impaired social memory in offspring. In the medial prefrontal cortex (mPFC), ZIKV-affected offspring mice exhibit excitation and inhibition imbalance and increased cortical activity. This could be explained by dysregulation of inhibitory neurons and synapses, and elevated neural activity input from mPFC-projecting ventral hippocampus (vHIP) neurons. We find structure alterations in the synaptic connections and pattern of vHIP innervation of mPFC neurons, leading to hyperconnectivity of the vHIP-mPFC pathway. Decreasing the activity of mPFC-projecting vHIP neurons with a chemogenetic strategy rescues social memory deficits in ZIKV offspring mice. Our studies reveal a hyperconnectivity of vHIP to mPFC projection driving social memory deficits in mice exposed to maternal inflammation by ZIKV.

Asymmetric Cell Division of Fibroblasts is An Early Deterministic Step to Generate Elite Cells during Cell Reprogramming.

Cell reprogramming is considered a stochastic process, and it is not clear which cells are prone to be reprogrammed and whether a deterministic step exists. Here, asymmetric cell division (ACD) at the early stage of induced neuronal (iN) reprogramming is shown to play a deterministic role in generating elite cells for reprogramming. Within one day, fibroblasts underwent ACD, with one daughter cell being converted into an iN precursor and the other one remaining as a fibroblast. Inhibition of ACD significantly inhibited iN conversion. Moreover, the daughter cells showed asymmetric DNA segregation and histone marks during cytokinesis, and the cells inheriting newly replicated DNA strands during ACD became iN precursors. These results unravel a deterministic step at the early phase of cell reprogramming and demonstrate a novel role of ACD in cell phenotype change. This work also supports a novel hypothesis that daughter cells with newly replicated DNA strands are elite cells for reprogramming, which remains to be tested in various reprogramming processes.

Organoids as Novel Models for Embryo Implantation Study.

In the last decade, organoids have become emerging novel models for biomedical research. Organoids are small, self-organized three-dimensional (3D) tissue cultures derived from stem cells that mimic certain tissues or organs. In reproductive medicine, researchers have generated numerous organoids including blastoid (blastocyst organoid), endometrial organoid, and trophoblast organoid. These organdies provide useful models for studying the embryo implantation mechanism through observation of cell differentiation, gene expression, and epigenetic profiles at the implantation stage. As in vitro tissue models, organoids could be coupled with many other frontier technologies such as gene editing and genomic sequencing. However, the main drawback of organoids is that they do not fully mimic their counterparts in vivo tissues. Furthermore, there is a consensus of research ethics on organoids that may limit the types of studies that scientists perform with. Nevertheless, all discoveries and efforts surrounding organoids still greatly benefit therapy development for reproductive clinics.

Loss of KDM4B exacerbates bone-fat imbalance and mesenchymal stromal cell exhaustion in skeletal aging.

Skeletal aging is a complex process, characterized by a decrease in bone formation, an increase in marrow fat, and stem cell exhaustion. Loss of H3K9me3, a heterochromatin mark, has been proposed to be associated with aging. Here, we report that loss of KDM4B in mesenchymal stromal cells (MSCs) exacerbated skeletal aging and osteoporosis by reducing bone formation and increasing marrow adiposity via increasing H3K9me3. KDM4B epigenetically coordinated ß-catenin/Smad1-mediated transcription by removing repressive H3K9me3. Importantly, KDM4B ablation impaired MSC self-renewal and promoted MSC exhaustion by inducing senescence-associated heterochromatin foci formation, providing a mechanistic explanation for stem cell exhaustion with aging. Moreover, while KDM4B was required for parathyroid hormone-mediated bone anabolism, KDM4B depletion accelerated bone loss and marrow adiposity induced by a high-fat diet. Our results suggest that the epigenetic rejuvenation and reversing bone-fat imbalance might be new strategies for preventing and treating skeletal aging and osteoporosis by activating KDM4B in MSCs.

Single-cell analysis of nonhuman primate preimplantation development in comparison to humans and mice.

BACKGROUND: Genetic programs underlying preimplantation development and early lineage segregation are highly conserved across mammals. It has been suggested that nonhuman primates would be better model organisms for human embryogenesis, but a limited number of studies have investigated the monkey preimplantation development. In this study, we collect single cells from cynomolgus monkey preimplantation embryos for transcriptome profiling and compare with single-cell RNA-seq data derived from human and mouse embryos. RESULTS: By weighted gene-coexpression network analysis, we found that cynomolgus gene networks have greater conservation with human embryos including a greater number of conserved hub genes than that of mouse embryos. Consistently, we found that early ICM/TE lineage-segregating genes in monkeys exhibit greater similarity with human when compared to mouse, so are the genes in signaling pathways such as LRP1 and TCF7 involving in WNT pathway. Last, we tested the role of one conserved pre-EGA hub gene, SIN3A, using a morpholino knockdown of maternal RNA transcripts in monkey embryos followed by single-cell RNA-seq. We found that SIN3A knockdown disrupts the gene-silencing program during the embryonic genome activation transition and results in developmental delay of cynomolgus embryos. CONCLUSION: Taken together, our study provided new insight into evolutionarily conserved and divergent transcriptome dynamics during mammalian preimplantation development.

Characterizing disease progression of nonalcoholic steatohepatitis in Leptin-deficient rats by integrated transcriptome analysis.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease threatening human health, yet no medicine is developed to treat this disease. In this study, we first discovered that Leptin mutant rats (LepΔI14/ΔI14) exhibit characteristic NASH phenotypes including steatosis, lymphocyte infiltration, and ballooning after postnatal week 16. We then examined NASH progression by performing an integrated analysis of hepatic transcriptome in Leptin-deficient rats from postnatal 4 to 48 weeks. Initially, simple steatosis in LepΔI14/ΔI14 rats were observed with increased expression of the genes encoding for rate-limiting enzymes in lipid metabolism such as acetyl-CoA carboxylase and fatty acid synthase. When NASH phenotypes became well developed at postnatal week 16, we found gene expression changes in insulin resistance, inflammation, reactive oxygen species and endoplasmic reticulum stress. As NASH phenotypes further progressed with age, we observed elevated expression of cytokines and chemokines including C-C motif chemokine ligand 2, tumor necrosis factor ɑ, interleukin-6, and interleukin-1ß together with activation of the c-Jun N-terminal kinase and nuclear factor-κB pathways. Histologically, livers in LepΔI14/ΔI14 rats exhibited increased cell infiltration of MPO+ neutrophils, CD8+ T cells, CD68+ hepatic macrophages, and CCR2+ inflammatory monocyte-derived macrophages associated with macrophage polarization from M2 to M1. Subsequent cross-species comparison of transcriptomes in human, rat, and mouse NASH models indicated that Leptin-deficient rats bear more similarities to human NASH patients than previously established mouse NASH models. Taken together, our study suggests that LepΔI14/ΔI14 rats are a valuable pre-clinical rodent model to evaluate NASH drug safety and efficacy.

Single-cell RNA sequencing reveals heterogeneous tumor and immune cell populations in early-stage lung adenocarcinomas harboring EGFR mutations.

Lung adenocarcinoma (LUAD) harboring EGFR mutations prevails in Asian population. However, the inter-patient and intra-tumor heterogeneity has not been addressed at single-cell resolution. Here we performed single-cell RNA sequencing (scRNA-seq) of total 125,674 cells from seven stage-I/II LUAD samples harboring EGFR mutations and five tumor-adjacent lung tissues. We identified diverse cell types within the tumor microenvironment (TME) in which myeloid cells and T cells were the most abundant stromal cell types in tumors and adjacent lung tissues. Within tumors, accompanied by an increase in CD1C+ dendritic cells, the tumor-associated macrophages (TAMs) showed pro-tumoral functions without signature gene expression of defined M1 or M2 polarization. Tumor-infiltrating T cells mainly displayed exhausted and regulatory T-cell features. The adenocarcinoma cells can be categorized into different subtypes based on their gene expression signatures in distinct pathways such as hypoxia, glycolysis, cell metabolism, translation initiation, cell cycle, and antigen presentation. By performing pseudotime trajectory, we found that ELF3 was among the most upregulated genes in more advanced tumor cells. In response to secretion of inflammatory cytokines (e.g., IL1B) from immune infiltrates, ELF3 in tumor cells was upregulated to trigger the activation of PI3K/Akt/NF-κB pathway and elevated expression of proliferation and anti-apoptosis genes such as BCL2L1 and CCND1. Taken together, our study revealed substantial heterogeneity within early-stage LUAD harboring EGFR mutations, implicating complex interactions among tumor cells, stromal cells and immune infiltrates in the TME.

Single-cell RNA cap and tail sequencing (scRCAT-seq) reveals subtype-specific isoforms differing in transcript demarcation.

The differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Gene isoforms allow a single gene diverse functions across different cell types, and isoform dynamics allow different functions over time. However, methods to efficiently identify and quantify RNA isoforms genome-wide in single cells are still lacking. Here, we introduce single cell RNA Cap And Tail sequencing (scRCAT-seq), a method to demarcate the boundaries of isoforms based on short-read sequencing, with higher efficiency and lower cost than existing long-read sequencing methods. In conjunction with machine learning algorithms, scRCAT-seq demarcates RNA transcripts with unprecedented accuracy. We identified hundreds of previously uncharacterized transcripts and thousands of alternative transcripts for known genes, revealed cell-type specific isoforms for various cell types across different species, and generated a cell atlas of isoform dynamics during the development of retinal cones.