A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

Centralized banking of human embryonic stem (hES) cells is an endeavor that can benefit individual research efforts and enhance international collaboration but is complicated by the fact that the science is rapidly evolving in an environment of heterogeneous laws, guidelines, and ethical standards. Written from the vantage point of regulatory professionals, this article provides an overview of the benefits of and challenges facing hESC banking enterprises in general with a focus on a global centralized banking effort.

Hematopoietic stem cells (HSCs) and an earlier wave of definitive erythroid/myeloid progenitors (EMPs) differentiate from hemogenic endothelial cells in the conceptus. EMPs can be generated in vitro from embryonic or induced pluripotent stem cells, but efforts to produce HSCs have largely failed. The formation of both EMPs and HSCs requires the transcription factor Runx1 and its non-DNA binding partner core binding factor β (CBFβ). Here we show that the requirements for CBFβ in EMP and HSC formation in the conceptus are temporally and spatially distinct. Panendothelial expression of CBFβ in Tek-expressing cells was sufficient for EMP formation, but was not adequate for HSC formation. Expression of CBFβ in Ly6a-expressing cells, on the other hand, was sufficient for HSC, but not EMP, formation. The data indicate that EMPs and HSCs differentiate from distinct populations of hemogenic endothelial cells, with Ly6a expression specifically marking the HSC-generating hemogenic endothelium.

Histone crotonylation is a non-acetyl histone lysine modification that is as widespread as acetylation. However, physiological functions associated with histone crotonylation remain almost completely unknown. Here we report that histone crotonylation is crucial for endoderm differentiation. We demonstrate that key crotonyl-coenzyme A (CoA)-producing enzymes are specifically induced in endodermal cells during differentiation of human embryonic stem cells (hESCs) in vitro and in mouse embryos, where they function to increase histone crotonylation and enhance endodermal gene expression. Chemical enhancement of histone crotonylation promotes endoderm differentiation of hESCs, whereas deletion of crotonyl-CoA-producing enzymes reduces histone crotonylation and impairs meso/endoderm differentiation in vitro and in vivo. Our study uncovers a histone crotonylation-mediated mechanism that promotes endodermal commitment of pluripotent stem cells, which may have important implications for therapeutic strategies against a number of human diseases.

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration in vitro, in stem cell models, and in vivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.

The exocrine pancreas, consisting of ducts and acini, is the site of origin of pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the genesis and progression of human pancreatic diseases, including PDAC, is limited because of challenges in maintaining human acinar and ductal cells in culture. Here we report induction of human pluripotent stem cells toward pancreatic ductal and acinar organoids that recapitulate properties of the neonatal exocrine pancreas. Expression of the PDAC-associated oncogene GNASR201C induces cystic growth more effectively in ductal than acinar organoids, whereas KRASG12D is more effective in modeling cancer in vivo when expressed in acinar compared with ductal organoids. KRASG12D, but not GNASR201C, induces acinar-to-ductal metaplasia-like changes in culture and in vivo. We develop a renewable source of ductal and acinar organoids for modeling exocrine development and diseases and demonstrate lineage tropism and plasticity for oncogene action in the human pancreas.

We report the direct reprogramming of both adult human fibroblasts and blood cells into induced neural plate border stem cells (iNBSCs) by ectopic expression of four neural transcription factors. Self-renewing, clonal iNBSCs can be robustly expanded in defined media while retaining multilineage differentiation potential. They generate functional cell types of neural crest and CNS lineages and could be used to model a human pain syndrome via gene editing of SCN9A in iNBSCs. NBSCs can also be derived from human pluripotent stem cells and share functional and molecular features with NBSCs isolated from embryonic day 8.5 (E8.5) mouse neural folds. Single-cell RNA sequencing identified the anterior hindbrain as the origin of mouse NBSCs, with human iNBSCs sharing a similar regional identity. In summary, we identify embryonic NBSCs and report their generation by direct reprogramming in human, which may facilitate insights into neural development and provide a neural stem cell source for applications in regenerative medicine.

Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.

During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling, we find that hESCs commit to exiting pluripotency unexpectedly early. We show that bone morphogenetic protein 4 (BMP4), an important differentiation trigger, induces a subset of early genes to mirror the sustained, bistable dynamics of upstream signaling. Induction of one of these genes, GATA3, drives differentiation in the absence of BMP4. Conversely, GATA3 knockout delays differentiation and prevents fast commitment to differentiation. We show that positive feedback at the level of the GATA3-BMP4 axis induces fast, irreversible commitment to differentiation. We propose that early commitment may be a feature of BMP-driven fate choices and that interlinked feedback is the molecular basis for an irreversible transition from pluripotency to differentiation.

Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

Calorie restriction (CR) extends life span and ameliorates age-related pathologies in most species studied, yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle after transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.

Achieving the promises of stem cell science to generate precise disease models and designer cell samples for personalized therapeutics will require harnessing pheno-genotypic cell-level data quantitatively and predictively in the lab and clinic. Those requirements could be met by developing a Big-Data-driven stem cell science strategy and community.

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.

High-definition vision in humans and nonhuman primates is initiated by cone photoreceptors located within a specialized region of the retina called the fovea. Foveal cone death is the ultimate cause of central blindness in numerous retinal dystrophies, including macular degenerative diseases. 3D retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) hold tremendous promise to model and treat such diseases. To achieve this goal, RO cones should elicit robust and intrinsic light-evoked electrical responses (i.e., phototransduction) akin to adult foveal cones, which has not yet been demonstrated. Here, we show strong, graded, repetitive, and wavelength-specific light-evoked responses from RO cones. The photoresponses and membrane physiology of a significant fraction of these lab-generated cones are comparable with those of intact ex vivo primate fovea. These results greatly increase confidence in ROs as potential sources of functional human cones for cell replacement therapies, drug testing, and in vitro models of retinal dystrophies.

Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.

Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state.