Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.

Trans-Ancestral Genetic Risk Factors for Treatment-Related Type 2 Diabetes Mellitus in Survivors of Childhood Cancer.

PURPOSE: Type 2 diabetes mellitus (T2D) is a prevalent long-term complication of treatment in survivors of childhood cancer, with marked racial/ethnic differences in burden. In this study, we investigated trans-ancestral genetic risks for treatment-related T2D. PATIENTS AND METHODS: Leveraging whole-genome sequencing data from the St Jude Lifetime Cohort (N = 3,676, 304 clinically ascertained cases), we conducted ancestry-specific genome-wide association studies among survivors of African and European genetic ancestry (AFR and EUR, respectively) followed by trans-ancestry meta-analysis. Trans-/within-ancestry replication including data from the Childhood Cancer Survivor Study (N = 5,965) was required for prioritization. Three external general population T2D polygenic risk scores (PRSs) were assessed, including multiancestry PRSs. Treatment risk effect modification was evaluated for prioritized loci. RESULTS: Four novel T2D risk loci showing trans-/within-ancestry replication evidence were identified, with three loci achieving genome-wide significance (P < 5 × 10-8). Among these, common variants at 5p15.2 (LINC02112), 2p25.3 (MYT1L), and 19p12 (ZNF492) showed evidence of modifying alkylating agent-related T2D risk in both ancestral groups, but showed disproportionately greater risk in AFR survivors (AFR odds ratios [ORs], 3.95-17.81; EUR ORs, 2.37-3.32). In survivor-specific RNA-sequencing data (N = 207), the 19p12 locus variant was associated with greater ZNF492 expression dysregulation after exposures to alkylators. Elevated T2D risks across ancestry groups were only observed with increasing values for multiancestry T2D PRSs and were especially increased among survivors treated with alkylators (top v bottom quintiles: ORAFR, 20.18; P = .023; OREUR, 13.44; P = 1.3 × 10-9). CONCLUSION: Our findings suggest therapy-related genetic risks contribute to the increased T2D burden among non-Hispanic Black childhood cancer survivors. Additional study of how therapy-related genetic susceptibility contributes to this disparity is needed.

Nilotinib-induced alterations in endothelial cell function recapitulate clinical vascular phenotypes independent of ABL1.

Nilotinib is a highly effective treatment for chronic myeloid leukemia but has been consistently associated with the development of nilotinib-induced arterial disease (NAD) in a subset of patients. To date, which cell types mediate this effect and whether NAD results from on-target mechanisms is unknown. We utilized human induced pluripotent stem cells (hiPSCs) to generate endothelial cells and vascular smooth muscle cells for in vitro study of NAD. We found that nilotinib adversely affects endothelial proliferation and migration, in addition to increasing intracellular nitric oxide. Nilotinib did not alter endothelial barrier function or lipid uptake. No effect of nilotinib was observed in vascular smooth muscle cells, suggesting that NAD is primarily mediated through endothelial cells. To evaluate whether NAD results from enhanced inhibition of ABL1, we generated multiple ABL1 knockout lines. The effects of nilotinib remained unchanged in the absence of ABL1, suggesting that NAD results from off- rather than on-target signaling. The model established in the present study can be applied to future mechanistic and patient-specific pharmacogenomic studies.

A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

Nutritional requirements of human induced pluripotent stem cells.

The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established the suitable non-basal medium components for hiPSC growth, we develop a simplified basal medium consisting of just 39 components, demonstrating that many ingredients of DMEM/F12 are either not essential or are at suboptimal concentrations. This new basal medium along with the supplement, which we call BMEM, enhances the growth rate of hiPSCs over DMEM/F12-based media, supports derivation of multiple hiPSC lines, and allows differentiation to multiple lineages. hiPSCs cultured in BMEM consistently have enhanced expression of undifferentiated cell markers such as POU5F1 and NANOG, along with increased expression of markers of the primed state and reduced expression of markers of the naive state. This work describes titration of the nutritional requirements of human pluripotent cell culture and identifies that suitable nutrition enhances the pluripotent state.

A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD , SMAD6 , and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2 + cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach. HIGHLIGHTS: Using network-based algorithm Mogrify, acoustic liquid handling, and high-content kinetic imaging cytometry we screened the effect of 4,960 unique transcription factor combinations. Using 24 patient-specific human fibroblast samples we identified the combination of MYOCD , SMAD6 , and TBX20 (MST) as the most successful direct reprogramming combination. MST cocktail results in reprogrammed cells with spontaneous contraction, cardiomyocyte-like calcium transients, and expression of cardiomyocyte associated genes.

Evaluation of multivalent H2 influenza pandemic vaccines in mice.

Subtype H2 Influenza A viruses were the cause of a severe pandemic in the winter of 1957. However, this subtype no longer circulates in humans and is no longer included in seasonal vaccines. As a result, individuals under 50years of age are immunologically naïve. H2 viruses persist in aquatic birds, which were a contributing source for the 1957 pandemic, and have also been isolated from swine. Reintroduction of the H2 via zoonotic transmission has been identified as a pandemic risk, so pre-pandemic planning should include preparation and testing of vaccine candidates against this subtype. We evaluated the immunogenicity of two inactivated, whole virus influenza vaccines (IVV) in mice: a monovalent IVV containing human pandemic virus A/Singapore/1/1957 (H2N2), and a multivalent IVV containing human A/Singapore/1/1957, avian A/Duck/HongKong/319/1978 (H2N2), and swine A/Swine/Missouri/2124514/2006 (H2N3) viruses. While both vaccines induced protective immunity compared to naïve animals, the multivalent formulation was advantageous over the monovalent in terms of level and breadth of serological responses, neutralization of infectious virus, and reduction of clinical disease and respiratory tissue replication in mice. Therefore, multivalent pandemic H2 vaccines containing diverse viruses from animal reservoirs, are a potential option to improve the immune responses in a pre-pandemic scenario where antigenic identity cannot be predicted.

Replication Capacity of Avian Influenza A(H9N2) Virus in Pet Birds and Mammals, Bangladesh.

Avian influenza A(H9N2) is an agricultural and public health threat. We characterized an H9N2 virus from a pet market in Bangladesh and demonstrated replication in samples from pet birds, swine tissues, human airway and ocular cells, and ferrets. Results implicated pet birds in the potential dissemination and zoonotic transmission of this virus.