The efficient generation of hematopoietic stem cells (HSCs) from human-induced pluripotent stem cells (iPSCs) holds great promise in personalized transplantation therapies. However, the derivation of functional and transplantable HSCs from iPSCs has had very limited success thus far.

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b-estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose-derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)-induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂-induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.

USP7, a ubiquitin-specific peptidase (USP), plays an important role in many cellular processes through its catalytic deubiquitination of various substrates. However, its nuclear function that shapes the transcriptional network in mouse embryonic stem cells (mESCs) remains poorly understood. We report that USP7 maintains mESC identity through both catalytic activity-dependent and -independent repression of lineage differentiation genes. Usp7 depletion attenuates SOX2 levels and derepresses lineage differentiation genes thereby compromising mESC pluripotency. Mechanistically, USP7 deubiquitinates and stabilizes SOX2 to repress mesoendodermal (ME) lineage genes. Moreover, USP7 assembles into RYBP-variant Polycomb repressive complex 1 and contributes to Polycomb chromatin-mediated repression of ME lineage genes in a catalytic activity-dependent manner. USP7 deficiency in its deubiquitination function is able to maintain RYBP binding to chromatin for repressing primitive endoderm-associated genes. Our study demonstrates that USP7 harbors both catalytic and noncatalytic activities to repress different lineage differentiation genes, thereby revealing a previously unrecognized role in controlling gene expression for maintaining mESC identity.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using oncogenic transcription factors. However, this method leads to genetic aberrations in iPSCs via unknown mechanisms, which may limit their clinical use. Here, we demonstrate that the supplementation of growth media with antioxidants reduces the genome instability of cells transduced with the reprogramming factors. Antioxidant supplementation did not affect transgene expression level or silencing kinetics. Importantly, iPSCs made with antioxidants had significantly fewer de novo copy number variations, but not fewer coding point mutations, than iPSCs made without antioxidants. Our results suggest that the quality and safety of human iPSCs might be enhanced by using antioxidants in the growth media during the generation and maintenance of iPSCs.

The reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, but also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established in parallel to the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. We show that OSKM induce expression of endodermal genes, leading to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to induced pluripotent stem cells, indicating that OSKM drive cells to two distinct cell fates during reprogramming.

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

As tendon stem/progenitor cells were reported to be rare in tendon tissues, tendons as vulnerable targets of sports injury possess poor self-repair capability. Human ESCs (hESCs) represent a promising approach to tendon regeneration. But their teno-lineage differentiation strategy has yet to be defined. Here, we report that force combined with the tendon-specific transcription factor scleraxis synergistically promoted commitment of hESCs to tenocyte for functional tissue regeneration. Force and scleraxis can independently induce tendon differentiation. However, force alone concomitantly activated osteogenesis, while scleraxis alone was not sufficient to commit, but augment tendon differentiation. Scleraxis synergistically augmented the efficacy of force on teno-lineage differentiation and inhibited the osteo-lineage differentiation by antagonized BMP signaling cascade. The findings not only demonstrated a novel strategy of directing hESC differentiation to tenocyte for functional tendon regeneration, but also offered insights into understanding the network of force, scleraxis and bmp2 controlling tendon-lineage differentiation.

Adults contracting Zika virus (ZIKV) typically exhibit mild symptoms, yet ZIKV infection of pregnant individuals can cause miscarriage or birth defects in their offspring. Many studies have focused on maternal-to-fetal ZIKV transmission via blood and placenta. Notably, however, ZIKV is also transmitted sexually, raising the possibility that ZIKV could infect the embryo shortly after fertilization, long before the placenta is established. Here, we evaluate the consequences of ZIKV infection in mouse embryos during the first few days of embryogenesis. We show that divergent strains of ZIKV can infect the fetal lineage and can cause developmental arrest, raising concern for the developmental consequences of sexual ZIKV transmission. This article has an associated 'The people behind the papers' interview.

Excessive pepsin can damage both normal laryngeal epithelial cells and laryngeal cancer (LC) cells. Heat shock protein 70 (HSP70) is closely related to pepsin. In this paper, we will explore the different significance of the regulatory role of HSP70 in endoplasmic reticulum stress (ERS) level in pepsin-treated laryngeal epithelial cells and LC cells.

Somatic cell reprogramming was first developed to create induced pluripotent stem (iPS) cells. Since that time, the highly dynamic and heterogeneous nature of the reprogramming process has come to be appreciated. Remarkably, a distinct type of stem cell, called induced extraembryonic endoderm (iXEN) stem cell, is also formed during reprogramming of mouse somatic cells by ectopic expression of the transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). The mechanisms leading somatic cells to adopt differing stem cell fates are challenging to resolve given that formation of either stem cell type is slow, stochastic, and rare. For these reasons, fluorescent gene expression reporters have provided an invaluable tool for revealing the path from the somatic state to pluripotency. However, no such reporters have been established for comparable studies of iXEN cell formation. In this study, we examined the expression of multiple fluorescent reporters, including Nanog, Oct4, and the endodermal genes, Gata4 and Gata6-alone and in combination, during reprogramming. We show that only simultaneous evaluation of Nanog and Gata4 reliably distinguishes iPS and iXEN cell colonies during reprogramming.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

In mice, pluripotent cells are thought to derive from cells buried inside the embryo around the 16-cell stage. Sox2 is the only pluripotency gene known to be expressed specifically within inside cells at this stage. To understand how pluripotency is established, we therefore investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4 (Pou5f1), highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1 and Tead4 Interestingly, the repression of premature Sox2 expression was sensitive to LATS kinase activity, even though LATS proteins normally do not limit activity of TEAD4, YAP1 and WWTR1 during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1, WWTR1 and TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the four-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10-15 years from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 FDA-approved compounds and clinical candidates, we identified 17 dose-responsive compounds with in vitro antiviral efficacy in human liver Huh7 cells and confirmed antiviral efficacy in human colon carcinoma Caco-2, human prostate adenocarcinoma LNCaP, and in a physiologic relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein classically found in secretory fluids, including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Articular cartilage injury and degeneration causing pain and loss of quality-of-life has become a serious problem for increasingly aged populations. Given the poor self-renewal of adult human chondrocytes, alternative functional cell sources are needed. Direct reprogramming by small molecules potentially offers an oncogene-free and cost-effective approach to generate chondrocytes, but has yet to be investigated. Here, we directly reprogrammed mouse embryonic fibroblasts into PRG4+ chondrocytes using a 3D system with a chemical cocktail, VCRTc (valproic acid, CHIR98014, Repsox, TTNPB, and celecoxib). Using single-cell transcriptomics, we revealed the inhibition of fibroblast features and activation of chondrogenesis pathways in early reprograming, and the intermediate cellular process resembling cartilage development. The in vivo implantation of chemical-induced chondrocytes at defective articular surfaces promoted defect healing and rescued 63.4% of mechanical function loss. Our approach directly converts fibroblasts into functional cartilaginous cells, and also provides insights into potential pharmacological strategies for future cartilage regeneration.

Fluorescent proteins and epitope tags can reveal protein localization in cells and animals, yet the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. Here, we present two strategies for higher throughput production of endogenous protein reporters in mice, focusing on the blastocyst model of development. Our first strategy makes use of a split fluorescent protein, mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest, was highly efficient in embryos, potentially obviating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. Our second strategy achieves in-frame knock-in of a relatively small protein tag, which provides high efficiency and higher sensitivity protein reporting. Together, these two approaches provide complementary advantages and enable broad downstream applications.

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFβ- and BMP-signaling, where inhibition of TGFβ- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFβ-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFβ- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.

Cancer and normal stem cells (SCs) share proliferative properties of self-renewal and expression of key transcription factors (TFs). Despite similar TF identities, the functional role of specific TFs responsible for retaining SC state has yet to be examined in cancer.