Deep-learning-based multi-class segmentation for automated, non-invasive routine assessment of human pluripotent stem cell culture status.

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into a variety of human tissue cells. They offer new opportunities for personalized medicine and drug screening. This requires large quantities of high quality hiPSCs, obtainable only via automated cultivation. One of the major requirements of an automated cultivation is a regular, non-invasive analysis of the cell condition, e.g. by whole-well microscopy. However, despite the urgency of this requirement, there are currently no automatic, image-processing-based solutions for multi-class routine quantification of this nature. This paper describes a method to fully automate the cell state recognition based on phase contrast microscopy and deep-learning. This approach can be used for in process control during an automated hiPSC cultivation. The U-Net based algorithm is capable of segmenting important parameters of hiPSC colony formation and can discriminate between the classes hiPSC colony, single cells, differentiated cells and dead cells. The model achieves more accurate results for the classes hiPSC colonies, differentiated cells, single hiPSCs and dead cells than visual estimation by a skilled expert. Furthermore, parameters for each hiPSC colony are derived directly from the classification result such as roundness, size, center of gravity and inclusions of other cells. These parameters provide localized information about the cell state and enable well based treatment of the cell culture in automated processes. Thus, the model can be exploited for routine, non-invasive image analysis during an automated hiPSC cultivation. This facilitates the generation of high quality hiPSC derived products for biomedical purposes.

The StemCellFactory: A Modular System Integration for Automated Generation and Expansion of Human Induced Pluripotent Stem Cells.

While human induced pluripotent stem cells (hiPSCs) provide novel prospects for disease-modeling, the high phenotypic variability seen across different lines demands usage of large hiPSC cohorts to decipher the impact of individual genetic variants. Thus, a much higher grade of parallelization, and throughput in the production of hiPSCs is needed, which can only be achieved by implementing automated solutions for cell reprogramming, and hiPSC expansion. Here, we describe the StemCellFactory, an automated, modular platform covering the entire process of hiPSC production, ranging from adult human fibroblast expansion, Sendai virus-based reprogramming to automated isolation, and parallel expansion of hiPSC clones. We have developed a feeder-free, Sendai virus-mediated reprogramming protocol suitable for cell culture processing via a robotic liquid handling unit that delivers footprint-free hiPSCs within 3 weeks with state-of-the-art efficiencies. Evolving hiPSC colonies are automatically detected, harvested, and clonally propagated in 24-well plates. In order to ensure high fidelity performance, we have implemented a high-speed microscope for in-process quality control, and image-based confluence measurements for automated dilution ratio calculation. This confluence-based splitting approach enables parallel, and individual expansion of hiPSCs in 24-well plates or scale-up in 6-well plates across at least 10 passages. Automatically expanded hiPSCs exhibit normal growth characteristics, and show sustained expression of the pluripotency associated stem cell marker TRA-1-60 over at least 5 weeks (10 passages). Our set-up enables automated, user-independent expansion of hiPSCs under fully defined conditions, and could be exploited to generate a large number of hiPSC lines for disease modeling, and drug screening at industrial scale, and quality.

MicroRNAs Engage in Complex Circuits Regulating Adult Neurogenesis.

The finding that the adult mammalian brain is still capable of producing neurons has ignited a new field of research aiming to identify the molecular mechanisms regulating adult neurogenesis. An improved understanding of these mechanisms could lead to the development of novel approaches to delay cognitive decline and facilitate neuroregeneration in the adult human brain. Accumulating evidence suggest microRNAs (miRNAs), which represent a class of post-transcriptional gene expression regulators, as crucial part of the gene regulatory networks governing adult neurogenesis. This review attempts to illustrate how miRNAs modulate key processes in the adult neurogenic niche by interacting with each other and with transcriptional regulators. We discuss the function of miRNAs in adult neurogenesis following the life-journey of an adult-born neuron from the adult neural stem cell (NSCs) compartment to its final target site. We first survey how miRNAs control the initial step of adult neurogenesis, that is the transition of quiescent to activated proliferative adult NSCs, and then go on to discuss the role of miRNAs to regulate neuronal differentiation, survival, and functional integration of the newborn neurons. In this context, we highlight miRNAs that converge on functionally related targets or act within cross talking gene regulatory networks. The cooperative manner of miRNA action and the broad target repertoire of each individual miRNA could make the miRNA system a promising tool to gain control on adult NSCs in the context of therapeutic approaches.

Notch/Hes signaling and miR-9 engage in complex feedback interactions controlling neural progenitor cell proliferation and differentiation.

Canonical Notch signaling has diverse functions during nervous system development and is critical for neural progenitor self-renewal, timing of differentiation and specification of various cell fates. A key feature of Notch-mediated self-renewal is its fluctuating activity within the neural progenitor cell population and the oscillatory expression pattern of the Notch effector Hes1 and its target genes. A negative feedback loop between Hes1 and neurogenic microRNA miR-9 was found to be part of this oscillatory clock. In a recent study we discovered that miR-9 expression is further modulated by direct binding of the Notch intracellular domain/RBPj transcriptional complex to the miR-9_2 promoter. In turn, miR-9 not only targets Hes1 but also Notch2 to attenuate Notch signaling and promote neuronal differentiation. Here, we discuss how the two interwoven feedback loops may provide an additional fail-save mechanism to control proliferation and differentiation within the neural progenitor cell population. Furthermore, we explore potential implications of miR-9-mediated regulation of Notch/Hes1 signaling with regard to neural progenitor homeostasis, patterning, timing of differentiation and tumor formation.

Reciprocal Regulation between Bifunctional miR-9/9(∗) and its Transcriptional Modulator Notch in Human Neural Stem Cell Self-Renewal and Differentiation.

Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9(∗) has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9(∗) decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9(∗) depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9(∗) via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9(∗)_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9(∗) and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells.

The role of microRNAs in human neural stem cells, neuronal differentiation and subtype specification.

The impressive neuronal diversity found within the nervous system emerges from a limited pool of neural progenitor cells that proceed through different gene expression programs to acquire distinct cell fates. Here, we review recent evidence indicating that microRNAs (miRNAs) are critically involved in conferring neural cell identities during neural induction, neuronal differentiation and subtype specification. Several studies have shown that miRNAs act in concert with other gene regulatory factors and genetic switches to regulate the spatial and temporal expression profiles of important cell fate determinants. So far, most studies addressing the role of miRNAs during neurogenesis were conducted using animal models. With the advent of human pluripotent stem cells and the possibility to differentiate these into neural stem cells, we now have the opportunity to study miRNAs in a human context. More insight into the impact of miRNA-based regulation during neural fate choice could in the end be exploited to develop new strategies for the generation of distinct human neuronal cell types.

Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

MicroRNA-based promotion of human neuronal differentiation and subtype specification.

MicroRNAs are key regulators of neural cell proliferation, differentiation and fate choice. Due to the limited access to human primary neural tissue, the role of microRNAs in human neuronal differentiation remains largely unknown. Here, we use a population of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from human embryonic stem cells to study the expression and function of microRNAs at early stages of human neural stem cell differentiation and neuronal lineage decision. Based on microRNA expression profiling followed by gain- and loss-of-function analyses in lt-NES cells and their neuronal progeny, we demonstrate that miR-153, miR-324-5p/3p and miR-181a/a contribute to the shift of lt-NES cells from self-renewal to neuronal differentiation. We further show that miR-125b and miR-181a specifically promote the generation of neurons of dopaminergic fate, whereas miR-181a inhibits the development of this neurotransmitter subtype. Our data demonstrate that time-controlled modulation of specific microRNA activities not only regulates human neural stem cell self-renewal and differentiation but also contributes to the development of defined neuronal subtypes.

Correction: MicroRNA-Based Promotion of Human Neuronal Differentiation and Subtype Specification.

[This corrects the article DOI: 10.1371/journal.pone.0059011.].