Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.

Background The efficacy and safety profiles of vaccines against SARS-CoV-2 in patients with cancer is unknown. We aimed to assess the safety and immunogenicity of the BNT162b2 (Pfizer–BioNTech) vaccine in patients with cancer. Methods For this prospective observational study, we recruited patients with cancer and healthy controls (mostly health-care workers) from three London hospitals between Dec 8, 2020, and Feb 18, 2021. Participants who were vaccinated between Dec 8 and Dec 29, 2020, received two 30 μg doses of BNT162b2 administered intramuscularly 21 days apart; patients vaccinated after this date received only one 30 μg dose with a planned follow-up boost at 12 weeks. Blood samples were taken before vaccination and at 3 weeks and 5 weeks after the first vaccination. Where possible, serial nasopharyngeal real-time RT-PCR (rRT-PCR) swab tests were done every 10 days or in cases of symptomatic COVID-19. The coprimary endpoints were seroconversion to SARS-CoV-2 spike (S) protein in patients with cancer following the first vaccination with the BNT162b2 vaccine and the effect of vaccine boosting after 21 days on seroconversion. All participants with available data were included in the safety and immunogenicity analyses. Ongoing follow-up is underway for further blood sampling after the delayed (12-week) vaccine boost. This study is registered with the NHS Health Research Authority and Health and Care Research Wales (REC ID 20/HRA/2031). Findings 151 patients with cancer (95 patients with solid cancer and 56 patients with haematological cancer) and 54 healthy controls were enrolled. For this interim data analysis of the safety and immunogenicity of vaccinated patients with cancer, samples and data obtained up to March 19, 2021, were analysed. After exclusion of 17 patients who had been exposed to SARS-CoV-2 (detected by either antibody seroconversion or a positive rRT-PCR COVID-19 swab test) from the immunogenicity analysis, the proportion of positive anti-S IgG titres at approximately 21 days following a single vaccine inoculum across the three cohorts were 32 (94%; 95% CI 81–98) of 34 healthy controls; 21 (38%; 26–51) of 56 patients with solid cancer, and eight (18%; 10–32) of 44 patients with haematological cancer. 16 healthy controls, 25 patients with solid cancer, and six patients with haematological cancer received a second dose on day 21. Of the patients with available blood samples 2 weeks following a 21-day vaccine boost, and excluding 17 participants with evidence of previous natural SARS-CoV-2 exposure, 18 (95%; 95% CI 75–99) of 19 patients with solid cancer, 12 (100%; 76–100) of 12 healthy controls, and three (60%; 23–88) of five patients with haematological cancers were seropositive, compared with ten (30%; 17–47) of 33, 18 (86%; 65–95) of 21, and four (11%; 4–25) of 36, respectively, who did not receive a boost. The vaccine was well tolerated; no toxicities were reported in 75 (54%) of 140 patients with cancer following the ...

Highlights d Cardiac fibroblasts and endothelial cells induce hiPSCcardiomyocyte maturation d CX43 gap junctions form between cardiac fibroblasts and cardiomyocytes d cAMP-pathway activation contributes to hiPSCcardiomyocyte maturation d Patient-derived hiPSC-cardiac fibroblasts cause arrhythmia in microtissues

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

To realize the therapeutic potential of human embryonic stem cells (hESCs), it is necessary to regulate their differentiation in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serumfree medium were aggregated by centrifugation to foster the formation of embryoid bodies (

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.

IntroductionIn vertebrate species, a prerequisite for the development of the primary germ layers is the commitment of primitive ectoderm (epiblast) cells to gastrulation. [3][4][5][6] In mammalian embryos, this process is accompanied by the formation of the primitive streak, a morphologic structure initiating at the prospective embryonic posterior. [6][7][8] In the mouse epiblast, cells ingressing through the streak emerge as either definitive endoderm or mesoderm, the latter including the progenitors of the hematopoietic system. 9 In the mouse, primitive streak cells are marked by expression of the transcription factor Mixl1 1,2 and mouse embryos deficient in Mixl1 display multiple defects in the formation of mesodermal and endodermal derived structures. 10 Consistent with this, more recent studies have confirmed that Mixl1 expression marks precursors of both mesoderm 11 and endoderm. 12 These latter studies took advantage of embryonic stem cells (ESCs) or mice in which one Mixl1 allele had been replaced by sequences encoding green fluorescent protein (GFP), facilitating the identification and isolation of viable GFP ϩ (Mixl1 ϩ ) primitive streak-like cells. Analysis of Mixl1 GFP/w mouse ESCs showed that a GFP ϩ (Mixl1 ϩ ) population present at differentiation days 3 and 4 contained hematopoietic precursors, 11 supporting previous data indicating that, in mouse embryos, such precursors arise directly from the primitive streak. 13 The majority of progenitors at this time were hemangioblasts, precursors with both hematopoietic and endothelial potential which, in the embryo, contribute to the primary vascular plexus and primitive erythropoiesis of the yolk sac. Thus, these progenitors as well as lineage restricted primitive erythroid precursors represent the first differentiated mesodermal derivatives that arise after the onset of gastrulation at embryonic day (E) 6.5. 9Because of the scarcity of examples, events surrounding gastrulation in the human have largely been inferred from comparative embryology, 8 a situation that has led to uncertainty surrounding the relationship between the first mesodermal like cells (mesoblasts) documented from postovulation day 13 onward, the appearance of hematopoietic cells, and the overt manifestation of the primitive streak, a structure that is first visible in embryos representing embryonic day 15 (E15). 8,[14][15][16] Differentiating human embryonic stem cells (HESCs) represent an experimental platform for dissecting the relationship between specific lineages and the early differentiation events surrounding formation of the primary germ layers. To examine the correlation between mesoderm formation in the human and the emergence of hematopoietic precursors, we targeted sequences encoding GFP to the MIXL1 locus using homologous recombination. We demonstrate that GFP fluorescence faithfully reported expression of the endogenous MIXL1 gene and that a mesodermal cell population defined by coexpression of GFP (MIXL1) and the platelet-derived growth factor receptor alpha (PDGFR␣) wa...

SummaryMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.