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Summary Several protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation., Graphical Abstract, Highlights • TNNI3 and TNNT2 proteins are present in day 25 hPSC-CMs • Commonly used reagents can lead to non-specific binding of anti-troponin antibodies • A fit-for-purpose workflow describes how to develop a flow cytometry protocol • A robust protocol for routine quality control testing was validated for hPSC-CMs, Waas and colleagues demonstrate pitfalls with popular antibodies and sample preparation conditions commonly used for the assessment of cardiomyocyte identity within differentiation cultures. By using a rigorous fit-for-purpose workflow, the authors developed and validated a comprehensive protocol to accurately assess cardiomyocyte identity within hPSC-CM cultures. The new protocol includes stepwise instructions to facilitate its implementation by experts and novices alike.

The development of mouse genetic tools has made a significant contribution to the understanding of skeletal and hematopoietic stem cell niches in bone marrow (BM). However, many experimental designs (e.g., selections of marker genes, target vector constructions, and choices of reporter murine strains) have unavoidable technological limitations and bias, which lead to experimental discrepancies, data reproducibility issues, and frequent data misinterpretation. Consequently, there are a number of conflicting views relating to fundamental biological questions, including origins and locations of skeletal and hematopoietic stem cells in the BM. In this report, we systematically unravel complicated data interpretations via comprehensive analyses of technological benefits, pitfalls, and challenges in frequently used mouse models and discuss their translational relevance to human stem cell biology. Particularly, we emphasize the important roles of using large human genomic data-informatics in facilitating genetic analyses of mouse models and resolving existing controversies in mouse and human BM stem cell biology., In this article, Chen and colleagues discuss technological challenges for genetic analysis of bone marrow stem cell niches. This article consists of a concise review, considerable perspectives, and in-depth data analysis. Importantly, the authors provide translational relevance through interrogating mouse genetic models with human genomic datasets. This review would aid to resolve data inconsistencies and their associated stem cell controversies.

Summary Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development., Highlights • Systematic comparison of three protocols for hPSC-pancreatic progenitor differentiation • Individual hPSC lines differentiate preferentially with different protocols • Shared transcriptomic and epigenomic signatures of hPSC-pancreatic progenitors • Molecular characterization of hPSC-pancreatic progenitors guides protocol refinement, Wesolowska-Andersen and colleagues present a comprehensive molecular and functional analysis of human PSC-derived pancreatic progenitors derived using three differentiation protocols. They define their shared transcriptomic and epigenomic signatures but also highlight significant differences between protocols, which are then used to guide protocol modifications to enhance further differentiation toward endocrine lineage.

Summary Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types., Graphical Abstract, Highlights • Core transcription factors (TFs) are predicted for >200 cell types/tissues • Predicted TFs for retinal pigment epithelial (RPE) cells can reprogram fibroblasts • These reprogrammed RPE-like cells are functionally similar to primary RPE • The sets of predicted factors may facilitate studies of control of cell identity, Small sets of core transcription factors establish gene expression programs that determine cellular function. Young, Lee, and colleagues have generated an atlas of candidate core transcription factors for over 200 human cell types. Candidate core transcription factors proved capable of reprogramming cells, suggesting this atlas may facilitate studies of transcriptional regulation and reprogramming for clinically important cell types.