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The generation of organoids and tissues with programmable cellular complexity, architecture and function would benefit from the simultaneous differentiation of human induced pluripotent stem cells (hiPSCs) into divergent cell types. Yet differentiation protocols for the overexpression of specific transcription factors typically produce a single cell type. Here we show that patterned organoids and bioprinted tissues with controlled composition and organization can be generated by simultaneously co-differentiating hiPSCs into distinct cell types via the forced overexpression of transcription factors, independently of culture-media composition. Specifically, we used such orthogonally induced differentiation to generate endothelial cells and neurons from hiPSCs in a one-pot system containing either neural or endothelial stem-cell-specifying media, and to produce vascularized and patterned cortical organoids within days by aggregating inducible-transcription-factor and wild-type hiPSCs into randomly pooled or multicore-shell embryoid bodies. Moreover, by leveraging multimaterial bioprinting of hiPSC inks without extracellular matrix, we generated patterned neural tissues with layered regions composed of neural stem cells, endothelium and neurons. Orthogonally induced differentiation of stem cells may facilitate the fabrication of engineered tissues for biomedical applications.

Introduction: Cardiovascular comorbidities may predispose to adverse outcomes in hospitalized patients with coronavirus disease 2019 (COVID-19). However, across the USA, the burden of cardiovascular comorbidities varies significantly. Whether clinical outcomes of hospitalized patients with COVID-19 differ between regions has not yet been studied systematically. Here, we report differences in underlying cardiovascular comorbidities and clinical outcomes of patients hospitalized with COVID-19 in Texas and in New York state. Methods: We established a multicenter retrospective registry including patients hospitalized with COVID-19 between March 15 and July 12, 2020. Demographic and clinical data were manually retrieved from electronic medical records. We focused on the following outcomes: mortality, need for pharmacologic circulatory support, need for mechanical ventilation, and need for hemodialysis. Univariate and multivariate logistic regression analyses were performed. Results: Patients in the Texas cohort (n = 296) were younger (57 vs. 63 years, p value 2 vs. 28.5 kg/m2, p = 0.015), and they had higher rates of diabetes mellitus (41 vs. 30%; p = 0.014). In contrast, patients in the New York state cohort (n = 218) had higher rates of coronary artery disease (19 vs. 10%, p = 0.005) and atrial fibrillation (11 vs. 5%, p = 0.012). Pharmacologic circulatory support, mechanical ventilation, and hemodialysis were more frequent in the Texas cohort (21 vs. 13%, p = 0.020; 30 vs. 12%, p < 0.001; and 11 vs. 5%, p = 0.009, respectively). In-hospital mortality was similar between the 2 cohorts (16 vs. 18%, p = 0.469). After adjusting for differences in underlying comorbidities, only the use of mechanical ventilation remained significantly higher in the participating Texas hospitals (odds ratios [95% CI]: 3.88 [1.23, 12.24]). Median time to pharmacologic circulatory support was 8 days (interquartile range: 2, 13.8) in the Texas cohort compared to 1 day (0, 3) in the New York state cohort, while median time to in-hospital mortality was 16 days (10, 25.5) and 7 days (4, 14), respectively (both p < 0.001). In-hospital mortality was higher in the late versus the early study phase in the New York state cohort (24 vs. 14%, p = 0.050), while it was similar between the 2 phases in the Texas cohort (16 vs. 15%, p = 0.741). Conclusions: Geographical differences, including practice pattern variations and the impact of disease burden on provision of health care, are important for the evaluation of COVID-19 outcomes. Unadjusted data may cause bias affecting future regulatory policies and proper allocation of resources.

Introduction: Although primarily a respiratory illness, COVID-19 frequently involves the cardiovascular system. Hypothesis: Accordingly, we assessed whether abnormal electrocardiographic findings, including cardiac dysrhythmias, ST/T wave changes, and QTc interval prolongation, predict adverse outcomes in patients with COVID-19. Methods: A multi-center retrospective study of hospitalized patients with COVID-19 was performed across 4 hospitals in Texas and 1 in New York. Initial and subsequent ECG findings among patients with uncomplicated disease course, patients with adverse clinical outcomes (need for vasopressors, mechanical ventilation or renal replacement therapy) and patients who died were analyzed. Univariable and multivariable Cox analyses were performed. Results: We identified 297 consecutive patients with COVID-19. Of these patients, 91% had available ECGs. Median heart rate on initial ECG was 92 bpm (IQR: 27), and median QTc interval was 442 msec (IQR: 40). Longer QTc interval on initial ECG was associated with adverse clinical outcomes (443 msec, IQR: 43) and death (457 msec, IQR: 52, vs 441.5 msec, IQR: 36; p=0.033). Cardiac dysrhythmias or ST/T wave changes were noted in subsequent ECGs of 46% of patients and were associated with adverse clinical outcomes (58%) or death (68% vs 33%; HR: 2.2, 95% CI: 1.3-3.6; p=0.002). Sixty-four patients (23%) developed a QTc interval >500msec during their hospitalization. In multivariable Cox analysis, history of coronary artery disease was independently associated with development of QTc>500msec (HR 2.1, 95% CI 1.0-4.4; p=0.047) while other baseline comorbidities were not. Patients with QTc>500msec were more commonly treated with azithromycin (91% vs 77%; p=0.048); no other differences in COVID-19 treatment were identified. In-hospital mortality among patients with QTc>500msec was 37% versus 13% among patients with shorter QTc intervals (HR: 2.6, 95% CI 1.5-4.5; p=0.001). Conclusions: Prolonged QTc interval on initial ECG of COVID-19 patients predicted adverse outcomes and death. Azithromycin was associated with development of QTc>500msec. Patients with a QTc interval >500msec had a 2.6 times higher risk for in-hospital mortality compared to patients with shorter QTc intervals.

Introduction: In a time span of 6 months, COVID-19 has infected more than 7 million and cost the lives of more than 400,000 people worldwide. Cardiovascular involvement is associated with adverse clinical outcomes. Hypothesis: Increased plasma troponin and brain natriuretic peptide (BNP) or NT-proB-type natriuretic peptide (NT-proBNP) levels are associated with the presence of underlying cardiovascular disease and predict increased in-hospital mortality. Methods: Here we present a multicenter observational study of patients diagnosed with COVID-19, who were hospitalized in 5 hospitals (4 in Texas and 1 in New York) between March 16 th and May 22 nd , 2020. Demographic and clinical characteristics were abstracted after patient’s discharge or death. To address the non-standard troponin and BNP/NT-proBNP assays across institutions we used the upper 99th percentile as the cut-off for abnormal on each test. Results: We identified 297 consecutive patients hospitalized with COVID-19. Troponin levels were available in 84% and BNP/NT-proBNP levels in 47% of patients. Troponin levels were increased in 14% and BNP/NT-proBNP in 46% of patients. In multivariate Cox analysis, history of coronary artery disease was independently associated with elevated troponin levels (Hazard Ratio [HR]: 5.24; 95% confidence interval [CI]: 1.37-19.97; p=0.015), while history of congestive heart failure was independently associated with elevated BNP/NT-proBNP levels (HR: 2.58; 95% CI: 1.05 6.31; p=0.038). Older age, history of hypertension, dyslipidemia, or stroke, were associated with elevated troponin or BNP/NT-proBNP levels only in the univariate analysis. Other demographic and clinical characteristics were not significantly associated with cardiac biomarker elevation. Collectively, elevated troponin or BNP/NT-proBNP were significantly associated with in-hospital mortality (HR: 3.47 95% CI: 1.90-6.35 and HR: 3.56; 95% CI: 1.44-8.77, respectively; both p Conclusions: Increased levels of cardiac biomarkers were associated with a 3-fold increase of in-hospital mortality among patients with COVID-19. Troponin or BNP/pro-BNP testing has a prognostic value and may guide treatment.

Simultaneous differentiation of human induced pluripotent stem cells (hiPSCs) into divergent cell types offers a pathway to achieving tailorable cellular complexity, patterned architecture, and function in engineered human organoids and tissues. Recent transcription factor (TF) overexpression protocols typically produce only one cell type of interest rather than the multitude of cell types and structural organization found in native human tissues. Here, we report an orthogonal differentiation platform for genomically programming stem cells, organoids and bioprinted tissues with controlled composition and organization. To demonstrate this platform, we orthogonally differentiated endothelial cells and neurons from hiPSCs in a one-pot system containing neural stem cell-specifying media. By aggregating inducible-TF and wildtype hiPSCs into pooled and multicore-shell embryoid bodies, we produced vascularized and patterned cortical organoids within days. Using multimaterial 3D bioprinting, we patterned 3D neural tissues from densely cellular, matrix-free stem cell inks that were orthogonally differentiated on demand into distinct layered regions composed of neural stem cells, endothelium, and neurons, respectively. Given the high proliferative capacity and patient-specificity of hiPSCs, our platform provides a facile route for programming cells and multicellular tissues for drug screening and therapeutic applications.

We report a biomanufacturing method for creating perfusable human tissues composed of stem cell–derived organ building blocks., Engineering organ-specific tissues for therapeutic applications is a grand challenge, requiring the fabrication and maintenance of densely cellular constructs composed of ~108 cells/ml. Organ building blocks (OBBs) composed of patient-specific–induced pluripotent stem cell–derived organoids offer a pathway to achieving tissues with the requisite cellular density, microarchitecture, and function. However, to date, scant attention has been devoted to their assembly into 3D tissue constructs. Here, we report a biomanufacturing method for assembling hundreds of thousands of these OBBs into living matrices with high cellular density into which perfusable vascular channels are introduced via embedded three-dimensional bioprinting. The OBB matrices exhibit the desired self-healing, viscoplastic behavior required for sacrificial writing into functional tissue (SWIFT). As an exemplar, we created a perfusable cardiac tissue that fuses and beats synchronously over a 7-day period. Our SWIFT biomanufacturing method enables the rapid assembly of perfusable patient- and organ-specific tissues at therapeutic scales.