Lineage Gene Expression Research Articles

Distinct dynamics of parental 5-hydroxymethylcytosine during human preimplantation development regulate early lineage gene expression

The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development.

LinRace: cell division history reconstruction of single cells using paired lineage barcode and gene expression data

Lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes in single cells, which allows for inference of cell lineage and cell types at the whole organism level. While most state-of-the-art methods for lineage reconstruction utilize only the lineage barcode data, methods that incorporate gene expressions are emerging. Effectively incorporating the gene expression data requires a reasonable model of how gene expression data changes along generations of divisions. Here, we present LinRace (Lineage Reconstruction with asymmetric cell division model), which integrates lineage barcode and gene expression data using asymmetric cell division model and infers cell lineages and ancestral cell states using Neighbor-Joining and maximum-likelihood heuristics. On both simulated and real data, LinRace outputs more accurate cell division trees than existing methods. With inferred ancestral states, LinRace can also show how a progenitor cell generates a large population of cells with various functionalities.

3D Enhancer-promoter networks provide predictive features for gene expression and coregulation in early embryonic lineages.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.

Zfp281 and Zfp148 control CD4 + T cell thymic development and T H 2 functions

How CD4 + lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by T helper cell type 2 (T H 2) effector cells. Genetic, single-cell, and spatial transcriptomic analyses showed that these factors promote the intrathymic CD4 + T cell differentiation of class II major histocompatibility complex (MHC II)–restricted thymocytes, including expression of the CD4 + lineage–committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promoted chromatin opening at and expression of T H 2 cytokine genes but not of the T H 2 lineage–determining transcription factor Gata3. We found that Zfp281 interacts with Gata3 and is recruited to Gata3 genomic binding sites at loci encoding Thpok and T H 2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4 + T cell development and T H 2 cell responses.

Abstract 14659: Muscle Oxygenation Responses to Acute Exercise and Cellular Biomarkers of Vascular Health in Veterans With Chronic Kidney Disease

Introduction: Increased susceptibility of endothelial dysfunction and skeletal muscle impairments suggest peripheral limitations as an explanation for diminished aerobic capacity in chronic kidney disease (CKD) patients. However, data on peripheral limitations in CKD patients is limited. Hypothesis: We hypothesized CKD patients would experience reduced muscle oxygenation responses to acute exercise and cellular evidence of declines in vascular health compared to those without CKD. Methods: Sixteen male Veterans (CKD: n=9, age 74±6 yrs, eGFR, 41.7±10 mL/min/1.73 m 2 ; non-CKD controls (CON): n=7, age, 67±8 yrs, eGFR, 75±10.6 mL/min/1.73 m 2 ) completed treadmill cardiopulmonary exercise testing via the Modified Bruce protocol. Oxygenation responses were obtained from the medial gastrocnemius using near-infrared spectroscopy. Blood samples were collected at rest and analyzed for colony-forming units (CFU), flow-cytometry of mononuclear cells (MNCs) focusing on endothelial lineage cells and gene expression of MNCs. Effect sizes between groups were determined using Hedge’s g (g). Results: At peak exercise, oxygen consumption (VO 2peak 19±5 vs 25.8±5.2 ml/kg/min -1 , g=1.2), heart rate (HR peak 132±21 vs 160±9 bpm, g=1.6), oxygen extraction (Δ[HHb] 14.8±10.8 vs 24.9±6.8 a.u., g=1.0), and change in total hemoglobin concentration (Δ[tHb] 8.9±5.1 vs 22.5±12.8 a.u., g=1.4) were lower in CKD compared to CON. At an exercise intensity of 3.4 METs, Δ[HHb] and Δ[tHb] were 42% and 54% lower (8.0±7.8 vs 13.8±8.8 a.u., g=0.6 and 4.9±4.1 vs 10.7±4.5 a.u., g=1.2) in CKD vs CON, despite no differences in oxygen consumption or heart rate. CD34 + 184 expression was 47% lower in CKD vs CON (0.2±0.04 vs 0.38±0.48 %, g=0.5). CFU was significantly inversely correlated with body mass index and waist circumference (r=-0.67, p=0.034; r=-0.65, p=0.041). Conclusions: Preliminary data from this ongoing study suggest oxygen extraction and capillary blood volume expansion are impaired during exercise in CKD patients when compared to those without CKD. CD34 + 184 expression was lower in CKD patients versus those without CKD, indicating poor hematopoietic stem cell migratory function in the CKD group. In addition, an inverse association between CFU and body habitus was observed.

Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.

Cardiac resident macrophages: key regulatory mediators in the aftermath of myocardial infarction.

Acute myocardial infarction (MI) is a prevalent and highly fatal global disease. Despite significant reduction in mortality rates with standard treatment regimens, the risk of heart failure (HF) remains high, necessitating innovative approaches to protect cardiac function and prevent HF progression. Cardiac resident macrophages (cMacs) have emerged as key regulators of the pathophysiology following MI. cMacs are a heterogeneous population composed of subsets with different lineage origins and gene expression profiles. Several critical aspects of post-MI pathophysiology have been shown to be regulated by cMacs, including recruitment of peripheral immune cells, clearance and replacement of damaged myocardial cells. Furthermore, cMacs play a crucial role in regulating cardiac fibrosis, risk of arrhythmia, energy metabolism, as well as vascular and lymphatic remodeling. Given the multifaceted roles of cMacs in post-MI pathophysiology, targeting cMacs represents a promising therapeutic strategy. Finally, we discuss novel treatment strategies, including using nanocarriers to deliver drugs to cMacs or using cell therapies to introduce exogenous protective cMacs into the heart.

Transcriptomics in serum and culture medium reveal shared and differential gene regulation in pathogenic and commensal Streptococcus suis.

Streptococcus suis colonizes the upper respiratory tract of healthy pigs at high abundance but can also cause opportunistic respiratory and systemic disease. Disease-associated S. suis reference strains are well studied, but less is known about commensal lineages. It is not known what mechanisms enable some S. suis lineages to cause disease while others persist as commensal colonizers, or to what extent gene expression in disease-associated and commensal lineages diverge. In this study we compared the transcriptomes of 21 S. suis strains grown in active porcine serum and Todd-Hewitt yeast broth. These strains included both commensal and pathogenic strains, including several strains of sequence type (ST) 1, which is responsible for most cases of human disease and is considered to be the most pathogenic S. suis lineage. We sampled the strains during their exponential growth phase and mapped RNA sequencing reads to the corresponding strain genomes. We found that the transcriptomes of pathogenic and commensal strains with large genomic divergence were unexpectedly conserved when grown in active porcine serum, but that regulation and expression of key pathways varied. Notably, we observed strong variation of expression across media of genes involved in capsule production in pathogens, and of the agmatine deiminase system in commensals. ST1 strains displayed large differences in gene expression between the two media compared to strains from other clades. Their capacity to regulate gene expression across different environmental conditions may be key to their success as zoonotic pathogens.

Abstract 2240: CK19 based molecular classification defined three distinct hepatocellular carcinoma subtypes with different cell lineage features and therapeutic vulnerabilities

Abstract Purpose: CK19 has been recognized as an important molecular marker for poor prognosis of Hepatocellular carcinoma (HCC), however, its molecular determinants and clinical implications is still remains a largely unmet challenge. We sought to identify molecular features and develop a CK19-based molecular classification model to provide new strategies for precise treatment of this deadly HCC. Experimental Design: Tumor and matched non-tumor liver specimens were collected from 116 patients who underwent partial hepatectomy for primary or recurrent HCC in Guangxi region. HCC patients were classified into two CK19 positive subtypes and one CK19 negative subtype based on CK19 related transcriptomic profiling. The tumor heterogeneity of three subtypes were further characterized by multiomics technology including whole-exome sequencing, bulk and single-cell RNA sequencing, whole genome methylation , and imaging mass cytometry (IMC). Subtype scoring were validated using the TCGA database and in vitro cell line. Results: We innovatively constructed an accurate classification scoring system for CK19 related HCC, which divided CK19 positive HCC into “Hepatic stem cell”(K1) and “Hepatic progenitor cell”(K2) two distinct subtypes with respective pathway and clinicopathological phenotypes . K1 is characterized by poor prognosis, and activation of Myc and proliferation pathway. Meanwhile, down-regulation of p53 pathway and low differentiation are also unique characteristics of K1, Compared with K1, K2 showed enrichment of pro-oncogene Ras pathway and high expression of inflammatory signals and immune cells. Moreover, these two CK19 positive subtypes have different cell lineage gene expression and they may come from different cell lineage types. K1 may come from CK19 negative HCC by epigenetic regulation but K2 may come from hepatic progenitor cells activated by inflammatory stimulation. Specifically, we identified K1 as a new poor prognosis stem cell subtype of CK19 positive HCC. K1 highly expressed tumor stem cell markers and displayed very low expression of androgen receptor AR, as well as activation of neural related signal pathway, which is ineffective for postoperative TACE treatment. Conclusion: These data suggest that lineage plasticity may contribute to heterogeneity of CK19 positive HCC, which in turn carry clinical implications regarding new treatment strategies for treatment of HCC with highly recurrence. Citation Format: Qiuyan Wang, Qinchen Lu, Yaobang Wang, Zhenxing Wang, Xi Wang, Yuting Tao, Bangde Xiang, Herry Yang. CK19 based molecular classification defined three distinct hepatocellular carcinoma subtypes with different cell lineage features and therapeutic vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2240.

Prediction and analysis of cis-regulatory elements in Dorsal and Ventral patterning genes of Tribolium castaneum and its comparison with Drosophila melanogaster.

Insect embryonic development and morphology are characterized by their anterior-posterior and dorsal-ventral (DV) patterning. In Drosophila embryos, DV patterning is mediated by a dorsal protein gradient which activates twist and snail proteins, the important regulators of DV patterning. To activate or repress gene expression, some regulatory proteins bind in clusters to their target gene at sites known as cis-regulatory elements or enhancers. To understand how variations in gene expression in different lineages might lead to different phenotypes, it is necessary to understand enhancers and their evolution. Drosophila melanogaster has been widely studied to understand the interactions between transcription factors and the transcription factor binding sites. Tribolium castaneum is an upcoming model animal which is catching the interest of biologists and the research on the enhancer mechanisms in the insect's axes patterning is still in infancy. Therefore, the current study was designed to compare the enhancers of DV patterning in the two insect species. The sequences of ten proteins involved in DV patterning of D. melanogaster were obtained from Flybase. The protein sequences of T. castaneum orthologous to those obtained from D. melanogaster were acquired from NCBI BLAST, and these were then converted to DNA sequences which were modified by adding 20kb sequences both upstream and downstream to the gene. These modified sequences were used for further analysis. Bioinformatics tools (Cluster-Buster and MCAST) were used to search for clusters of binding sites (enhancers) in the modified DV genes. The results obtained showed that the transcription factors in Drosophila melanogaster and Tribolium castaneum are nearly identical; however, the number of binding sites varies between the two species, indicating transcription factor binding site evolution, as predicted by two different computational tools. It was observed that dorsal, twist, snail, zelda, and Supressor of Hairless are the transcriptionfactorsresponsible for the regulation of DV patterning in the two insect species.

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EμB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation.

How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2and Isl1, including those dependent on Atoh7, and uncover the sequential and combinatorial interactions of these factors with the epigenetic landscape to control gene expression along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs).

Disease-associated oligodendrocyte signatures in neurodegenerative disease: the known and unknown.

Disease-associated oligodendrocyte signatures in neurodegenerative disease: the known and unknown.

SPI1 is a prognostic biomarker of immune infiltration and immunotherapy efficacy in clear cell renal cell carcinoma

BackgroundSpi-1 proto-oncogene (SPI1), which encodes an ETS-domain transcription factor, can activate gene expression in myeloid and lymphoid lineages. The role of SPI1 in the tumor immune microenvironment in clear cell renal cell carcinoma (ccRCC) remains unknown. In this study, we investigated the possible role of SPI1 in ccRCC using an independent cohort and a comprehensive bioinformatics analysis.Materials and methodsQuantitative real-time PCR, western blot and immunohistochemistry assays were used to compare the SPI1 expression levels between ccRCC tissues and normal tissues, analyze the relationships between SPI1 and CD68, CD8, CD4 expression levels, and explore the link between SPI1 and the efficacy of immunotherapy in our cohort. Tumor Immune Estimation Resource, UALCAN, cBioPortal, TISIDB database, and LinkedOmics database were used in our study.ResultsSPI1 expression level was higher in ccRCC bulk tissues than in normal bulk tissues. SPI1 was an independent prognostic factor for poor overall survival and progression-free survival in patients with ccRCC. SPI1 expression was strongly related to the infiltration of immune cells and immune-related molecules. SPI1 was more highly expressed in tumor-infiltrating immune cells rather than in cancer cells. Non-responders to immunotherapy against ccRCC were more likely to express higher SPI1 levels than responders. Genes co-expressed with SPI1 primarily correlated with immune-related pathways.ConclusionsSPI1 expression in tumor bulk tissues is associated with disease progression and poor prognosis, as well as high expression levels of immune markers and infiltration of immune cells. SPI1 can be used as a prognostic biomarker to monitor and evaluate immunotherapy efficacy.

Abstract B003: Inactivation mutations of Kmt2c/d license a molecular “field effect” and prime the urothelium for tumorigenesis

Abstract Urothelial carcinoma (UC) is widely recognized to arise from a “field” of precancerous but histologically normal urothelium (HNU). The molecular mechanism that licenses the “field effect” remains elusive. Recent studies revealed prevalent KMT2C and KMT2D loss-of-function (LOF) mutations in HNU and cancer adjacent urothelium, suggesting their potential involvement in UC initiation. Here, we demonstrated that knockout (KO) of Kmt2c and/or Kmt2d in murine urothelial cells induced drastic alterations of cellular states by single cell RNA analysis, but was insufficient to induce robust histological changes. Kmt2c/d loss enhanced organoid formation efficiency, induced epithelial-mesenchymal transition (EMT), and impaired urothelial differentiation, indicating the augmented lineage plasticity after Kmt2c/d KO. Additionally, we identified that Kmt2c/d KO induced a pre-tumorigenic transcriptome with increased enrichments of gene sets associated with inflammation and decreased enrichments of gene sets associated with differentiation. Consequently, loss of Kmt2c/d sensitized urothelial cells to common oncogenic mutations to initiate UC in mouse models. We further observed that KO of Kmt2c/d increased tumorigenic susceptibility in carcinogen-induced UC model. Mechanistically, we observed decreased H3K4me1, H3K27Ac histone marks and decreased enhancer RNA production at the majority of enhancers that correlate with downregulation of urothelial specific lineage gene expression after Kmt2c/d KO. Furthermore, we observed increased Menin deposition on promoters of up-regulated genes. Blockade of Menin-KMT2A complex by small molecule inhibitor partially rescued the EMT and basal differentiation induced by Kmt2c/d KO. Together, our data posit that Kmt2c/d represents a key molecular determinant and their functional loss licenses a molecular “field effect” which primes the urothelium for oncogenic transformation. Citation Format: Naitao Wang, Mohini R. Pachai, Dan Li, Cindy Lee, Sarah Warda, Guojia Xie, Cheng Qian, Wai Pung E. Wong, Juan Yan, Wenhuo Hu, Alison Smith, Kai Ge, Sarat Chandarlapaty, Gopakumar V. Iyer, Jonathan E. Rosenberg, David B. Solit, Hikmat A. AI-Ahmadie, Ping Chi, Yu Chen. Inactivation mutations of Kmt2c/d license a molecular “field effect” and prime the urothelium for tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B003.

Generation and integration of hair follicle-primed spheroids in bioengineered skin constructs

Skin is a complex organ made up of different cell layers, appendages, connective tissues, and immune repertoires. These different components interact extensively to maintain the overall functions of the integumentary system. In particular, appendages such as hair follicles critically contribute to the skin’s function in thermoregulation, sensory perception, and homeostatic regeneration. Despite a strong need for better skin regenerative therapeutics, efforts to bio-engineer highly functional appendage-containing human reconstituted skin in vitro have not yielded much success. Here, we report methods in generating and incorporating hair follicle-primed heterotypic spheroids into epidermal-dermal skin constructs that induced invaginating outgrowths with follicle-like organization and lineage gene expression. By co-culturing epithelial keratinocytes (KCs) with dermal papilla (DP) cells in low attachment plates, we established the media and culture conditions that best supported the viability, signalling and remodelling of the cell aggregates to form 3D KC-DP spheroids with the expression of both DP inductiveness and hair follicle lineage genes. We show that long-term growth and maturation of KC cells in these spheroids was supported by incorporation into epidermal-dermal constructs but not in scaffold-less media. When cultured, the bio-fabricated constructs developed invaginations from the integrated spheroids with follicle-forming potential. The generation of these constructs is a step towards the development of functional hair-bearing skin mimetics.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

TedSim: temporal dynamics simulation of single-cell RNA sequencing data and cell division history.

Recently, lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes, which allows for the reconstruction of the cell division tree and makes it possible to reconstruct ancestral cell types and trace the origin of each cell type. Meanwhile, trajectory inference methods are widely used to infer cell trajectories and pseudotime in a dynamic process using gene expression data of present-day cells. Here, we present TedSim (single-cell temporal dynamics simulator), which simulates the cell division events from the root cell to present-day cells, simultaneously generating two data modalities for each single cell: the lineage barcode and gene expression data. TedSim is a framework that connects the two problems: lineage tracing and trajectory inference. Using TedSim, we conducted analysis to show that (i) TedSim generates realistic gene expression and barcode data, as well as realistic relationships between these two data modalities; (ii) trajectory inference methods can recover the underlying cell state transition mechanism with balanced cell type compositions; and (iii) integrating gene expression and barcode data can provide more insights into the temporal dynamics in cell differentiation compared to using only one type of data, but better integration methods need to be developed.

Differentiation of Human Pluripotent Stem Cells into Pancreatic Beta-cell Precursors in a 2D Culture System.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,however,with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell's physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

Reversion Mosaicism in Primary Immunodeficiency Diseases.

Reversion mosaicism has been reported in an increasing number of genetic disorders including primary immunodeficiency diseases. Several mechanisms can mediate somatic reversion of inherited mutations. Back mutations restore wild-type sequences, whereas second-site mutations result in compensatory changes. In addition, intragenic recombination, chromosomal deletions, and copy-neutral loss of heterozygosity have been demonstrated in mosaic individuals. Revertant cells that have regained wild-type function may be associated with milder disease phenotypes in some immunodeficient patients with reversion mosaicism. Revertant cells can also be responsible for immune dysregulation. Studies identifying a large variety of genetic changes in the same individual further support a frequent occurrence of reversion mosaicism in primary immunodeficiency diseases. This phenomenon also provides unique opportunities to evaluate the biological effects of restored gene expression in different cell lineages. In this paper, we review the recent findings of reversion mosaicism in primary immunodeficiency diseases and discuss its clinical implications.