Stem Cell Biology Research Articles

A systematic review on the effects of ROCK inhibitors on proliferation and/or differentiation in human somatic stem cells: A hypothesis that ROCK inhibitors support corneal endothelial healing via acting on the limbal stem cell niche.

Rho kinase inhibitors (ROCKi) have attracted growing multidisciplinary interest, particularly in Ophthalmology where the question as to how they promote corneal endothelial healing remains unresolved. Concurrently, stem cell biology has rapidly progressed in unravelling drivers of stem cell (SC) proliferation and differentiation, where mechanical niche factors and the actin cytoskeleton are increasingly recognized as key players. There is mounting evidence from the study of the peripheral corneal endothelium that supports the likelihood of an internal limbal stem cell niche. The possibility that ROCKi stimulate the endothelial SC niche has not been addressed. Furthermore, there is currently a paucity of data that directly evaluates whether ROCKi promotes corneal endothelial healing by acting on this limbal SC niche located near the transition zone. Therefore, we performed a systematic review examining the effects ROCKi on the proliferation and differentiation of human somatic SC, to provide insight into its effects on various human SC populations. An appraisal of electronic searches of four databases identified 1 in vivo and 58 in vitro studies (36 evaluated proliferation while 53 examined differentiation). Types of SC studied included mesenchymal (n=32), epithelial (n=11), epidermal (n=8), hematopoietic and other (n=8). The ROCK 1/2 selective inhibitor Y-27632 was used in almost all studies (n=58), while several studies evaluated ≥2 ROCKi (n=4) including fasudil, H-1152, and KD025. ROCKi significantly influenced human somatic SC proliferation in 81% of studies (29/36) and SC differentiation in 94% of studies (50/53). The present systemic review highlights that ROCKi are influential in regulating human SC proliferation and differentiation, and provides evidence to support the hypothesis that ROCKi promotes corneal endothelial division and maintenance via acting on the inner limbal SC niche.

Interdisciplinary approaches to brain organoid biology

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used as materials for regenerative medicine and for modeling development and disease because of their pluripotency to differentiate into all cell types of the body. Recently, organoid research has attracted considerable attention as a constructive approach to reconstitute various tissues from ESCs or iPSCs in three-dimensional culture in vitro. Organoids can provide sophisticated in vitro models because of their ability to partially recapitulate different cell types of living tissues and their functions. However, given their complexity, conventional analyses of stem cell biology are insufficient for evaluating organoid performance, limiting basic research and clinical translation. In recent years, elucidating diverse and complex biological phenomena by integrating stem cell biology with other research fields has become feasible. In this review, we focus on brain organoids with some representative examples of interdisciplinary research using machine learning, genetic and viral engineering, and optical imaging, as well as findings obtained from such research. Furthermore, we will discuss the potential applications and future perspectives of interdisciplinary research in organoid biology.

Cardiac cell sheet engineering for regenerative medicine and tissue modeling.

Stem cell biology and tissue engineering are essential techniques for cardiac tissue construction. We have succeeded in fabricating human cardiac tissue using the mass production technology of human iPS cell-derived cardiomyocytes and cell sheet engineering, and we are developing regenerative medicine and tissue models to apply this tissue to heart disease research. Cardiac tissue fabrication and tissue functional evaluation technologies for contractile and electrophysiological function are indispensable, which lead to the functional improvement of bioengineered human cardiac tissue.

Whole Cell Patch Clamp Electrophysiology in Human Neuronal Cells.

Whole cell patch clamp recording techniques are commonly used to assay membrane excitability, ion channel function, and synaptic activity in neurons. However, assaying these functional properties of human neurons remains difficult because of the difficulty in obtaining human neuronal cells. Recent advents in stem cell biology, especially the development of the induced pluripotent stem cells, made it possible to generate human neuronal cells in both 2-dimensional (2D) monolayer cultures and 3D brain-organoid cultures. Here, we describe the whole cell patch clamp methods of recording neuronal physiology from human neuronal cells.

The centrality of proteoglycan research: Expect the unexpected

The centrality of proteoglycan research: Expect the unexpected

Biological Functions of the KDM2 Family of Histone Demethylases.

The histone lysine demethylase 2 (KDM2) family of α-Ketoglutarate-Fe++-dependent dioxygenases were the first Jumonji-domain-containing proteins reported to harbor demethylase activity. This landmark discovery paved the way for the characterization of more than 25 enzymes capable of demethylating lysine residues on histones-an epigenetic modification previously thought to be irreversible. The KDM2 family is comprised of KDM2A and KDM2B which share significant structural similarities and demethylate lysine 36 on histone H3. However, they exert distinct cellular functions and are frequently deregulated in a broad spectrum of human cancers. With the advent of next generation sequencing and development of genetically engineered mouse models, it was shown that KDM2A and KDM2B play critical roles in stem cell biology, somatic cell reprograming, and organismal development by regulating cell fate and lineage commitment decisions. Thus, understanding the biochemistry and elucidating the context-dependent function of these enzymes is an emerging new frontier for the development of small molecule inhibitors to treat cancer and other diseases.

Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.

The coordinated interaction between the intestinal epithelium and immune cells is required to maintain proper barrier function and mucosal host defenses to the harsh external environment of the gut lumen. Complementary to in vivo models, there is a need for practical and reproducible in vitro models that employ primary human cells to confirm and advance our understanding of mucosal immune responses under physiologic and pathophysiologic conditions. Here we describe the methods to co-culture human intestinal stem cell-derived enteroids grown as confluent monolayers on permeable supports with primary human innate immune cells (e.g., monocyte-derived macrophages and polymorphonuclear neutrophils). This co-culture model reconstructs the cellular framework of the human intestinal epithelial-immune niche with distinct apical and basolateral compartments to recreate host responses to luminal and submucosal challenges, respectively. Enteroid-immune co-cultures enable multiple outcome measures to interrogate important biological processes such as epithelial barrier integrity, stem cell biology, cellular plasticity, epithelial-immune cellscrosstalk, immune cell effector functions, changes in gene expression (i.e., transcriptomic, proteomic, epigenetic), and host-microbiome interactions.

Drug-resistant Stem Cell Models for the Hormone-responsive Luminal A Breast Cancer

Background: Progression of early stage breast cancer to advanced stage metastatic disease represents a major cause of death in women. The Luminal A breast cancer subtype exhibits acceptable response to Chemo-endocrine and targeted therapy. However, these treatment options are associated with intrinsic/acquired therapy resistance and emergence chemo-resistant cancer initiating stem cell population, and resultant progression to advanced stage metastatic disease. These limitations emphasize an unmet need for the development of reliable models for cancer stem cells that facilitate identification of efficacious therapeutic alternatives. Documented human consumption, low systemic toxicity, preclinical cancer growth inhibitory efficacy and stem cell targeting efficacy of natural products, such as dietary phytochemicals and nutritional herbs, provide mechanistic leads for these agents as testable therapeutic alternatives. Objectives: The objectives of the present review are to i.) Provide a systematic discussion of published evidence relevant conceptual background of conventional/targeted therapy and nutritional herbs as testable alternatives, ii.) Growth inhibitory efficacy of nutritional herbs in a cellular model for the Luminal A breast cancer, iii.) Breast cancer stem cell biology and stem cell models for therapy-resistant breast cancer, and iv.) Future research directions. Conclusions: Collectively, all the elements discussed in the present review validate mechanism-based experimental approaches to identify and prioritize potential therapeutic alternatives. Future Research: This review provides a rationale for investigations on patient-derived tumor samples that may minimize extrapolation of the preclinical data for their clinical relevance and translatability.

Perspectives: Approaches for Studying Livestock Spermatogonia.

At present, the knowledge base on characteristics and biology of spermatogonia in livestock is limited in comparison to rodents, yet the importance of studying these cells for comparative species analysis and enhancing reproductive capacity in food animals is high. Previous studies have established that although many core attributes of organ physiology and mechanisms governing essential cellular functions are conserved across eutherians, significant differences exist between mice and higher order mammals. In this chapter, we briefly discuss distinguishing aspects of testicular anatomy and the spermatogenic lineage in livestock and critical considerations for studying spermatogonial stem cell biology in these species.

Deep learning models will shape the future of stem cell research.

Our ability to understand and control stem cell biology is being augmented by developments on two fronts, our ability to collect more data describing cell state and our capability to comprehend these data using deep learning models. Here we consider the impact deep learning will have in the future of stem cell research. We explore the importance of generating data suitable for these methods, the requirement for close collaboration between experimental and computational researchers, and the challenges we face to do this fairly and effectively. Achieving this will ensure that the resulting deep learning models are biologically meaningful and computationally tractable.

Primary Cultures of Spermatogonia and Testis Cells.

Spermatogonial stem cells (SSCs) maintain adult spermatogenesis in mammals by undergoing self-renewal and differentiation into spermatozoa. In order to study the biology of SSCs as related to spermatogenesis, an in vitro, long-term expansion system of SSCs constitutes an ideal tool. In this chapter, we describe a robust culture system for mouse and rat SSCs in vitro. In the presence of GDNF, GFRα1, and bFGF, SSCs maintained on STO feeder layers with serum-free medium continuously proliferate for over 6months. Complete spermatogenesis in infertile recipient mice can be attained following transplantation of the cultured mouse and rat SSCs. Using the in vitro SSC culture systems, elucidation of stem cell biology can be advanced that significantly advances our understanding of spermatogenesis and male fertility.

RNA Modifications in Cancer Stem Cell Biology.

Post-transcriptional regulation of gene expression shapes the cell state both in health and disease. RNA modifications-especially N6-methyladenosine (m6A)-have recently emerged as key players in RNA processing that depends on a sophisticated interplay between proteins of the RNA modification machinery. Importantly, the RNA epitranscriptome becomes dysregulated in cancer and promotes cancer-associated gene expression programs as well as cancer cell adaptation to the tumor microenvironment. At the top of the tumor hierarchy, cancer stem cells (CSCs) are master regulators of tumorigenesis and resistance to therapeutic intervention. Therefore, defining how RNA modifications influence the CSC state is of great importance for cancer drug development. In this chapter, we summarize the current knowledge of the roles of RNA modifications in shaping the CSC state and driving gene expression programs that confer stem-like properties to CSCs, promote CSC adaptation to the local microenvironment, and endow CSCs with metastatic potential and drug resistance.

Gastric Stem Cell Biology and Helicobacter pylori Infection.

Helicobacter pylori colonizes the human gastric mucosa and persists lifelong. An interactive network between the bacteria and host cells shapes a unique microbial niche within gastric glands that alters epithelial behavior, leading to pathologies such as chronic gastritis and eventually gastric cancer. Gland colonization by the bacterium initiates aberrant trajectories by inducing long-term inflammatory and regenerative gland responses, which involve various specialized epithelial and stromal cells. Recent studies using cell lineage tracing, organoids and scRNA-seq techniques have significantly advanced our knowledge of the molecular "identity" of epithelial and stromal cell subtypes during normal homeostasis and upon infection, and revealed the principles that underly stem cell (niche) behavior under homeostatic conditions as well as upon H. pylori infection. The activation of long-lived stem cells deep in thegastric glands has emerged as a key prerequisite of H. pylori-associated gastric site-specific pathologies such as hyperplasia in the antrum, and atrophy or metaplasia in the corpus, that are considered premalignant lesions. In addition to altering the behaviour of bona fide stem cells, injury-driven de-differentiation and trans-differentation programs, such as "paligenosis", subsequently allow highly specialized secretory cells to re-acquire stem cell functions, driving gland regeneration. This plastic regenerative capacity of gastric glands is required to maintain homeostasis and repair mucosal injuries. However, these processes are co-opted in the context of stepwise malignant transformation in chronic H. pylori infection, causing the emergence, selection and expansion of cancer-promoting stem cells.

Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis

Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis

METHODS OF DIFFERENTIATION FROM PLURIPOTENT STEM CELLS TO NEURAL CELLS

İnsan embriyonik kök hücreleri, embriyoların erken
 blastokist evresindeki iç hücre kütlesinden türetilen
 hücrelerdir. Pluripotent özellikte olan bu hücreler, uygun
 koşullar altında fonksiyonel nöronlara ve farklı
 tipte sinir hücrelerine farklılaştırılabilmektedir. Ancak
 bu alandaki en büyük zorluklardan biri, yenilenebilir,
 kültürü kolay, nöral soylara bağlı nöral prekürsör
 hücre popülasyonu oluşturmaktır. Bu nedenle, insan
 embriyonik kök hücrelerini prekürsör hücrelere en
 uygun şekilde farklılaştırmak, bunların kendi kendini
 yenileyen bir popülasyon olarak devam etmesi ve
 farklı bölgelerdeki sinir hücre tiplerini saf bir popülasyon
 şeklinde üretmek için kritik öneme sahiptir. Hücre
 sinyalleri ve bunlarla ilişkili moleküller de bu olaylarda
 önemli bir rol oynamaktadır. Nöral prekürsör hücrelerinin
 üretilmesi için kök hücre biyolojisinin ve nöral
 hücrelere farklılaşmada rol oynayan önemli yolakların
 daha iyi anlaşılması gerekmektedir. Bu derlemede
 kök hücrelerden nöral hücrelere farklılaştırma yöntemlerine
 ve bu süreçte önemli olan sinyal yolaklarına
 ve moleküllere odaklanılmaktadır.

ISAnalytics enables longitudinal and high-throughput clonal tracking studies in hematopoietic stem cell gene therapy applications.

Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies.

MITOCHONDRIAL METABOLIC CHECKPOINT, STEM CELL AGING AND REJUVENATION

Abstract Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in stem cell biology highlight a mitochondrial metabolic checkpoint that is essential for stem cells to return to the quiescent state. As quiescent stem cells enter the cell cycle, mitochondrial biogenesis is induced and mitochondrial stress is increased. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow stem cells to exit the cell cycle and return to quiescence. These processes are critically regulated by several sirtuin family members, which are NAD+-dependent deacetylases. Because loss of stem cell quiescence results in the depletion of the stem cell pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in stem cells will increase our understanding of tissue homeostasis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions.

Notch signaling sculpts the stem cell niche.

Adult stem cells depend on their niches for regulatory signaling that controls their maintenance, division, and their progeny differentiation. While communication between various types of stem cells and their niches is becoming clearer, the process of stem cell niche establishment is still not very well understood. Model genetic organisms provide simplified systems to address various complex questions, for example, how is a stem cell niche formed? What signaling cascades induce the stem cell niche formation? Are the mechanisms of stem cell niche formation conserved? Notch signaling is an evolutionarily conserved pathway first identified in fruit flies, crucial in fate acquisition and spatiotemporal patterning. While the core logic behind its activity is fairly simple and requires direct cell-cell interaction, it reaches an astonishing complexity and versatility by combining its different modes of action. Subtleties such as equivalency between communicating cells, their physical distance, receptor and ligand processing, and endocytosis can have an effect on the way the events unfold, and this review explores some important general mechanisms of action, later on focusing on its involvement in stem cell niche formation. First, looking at invertebrates, we will examine how Notch signaling induces the formation of germline stem cell niche in male and female Drosophila. In the developing testis, a group of somatic gonadal precursor cells receive Delta signals from the gut, activating Notch signaling and sealing their fate as niche cells even before larval hatching. Meanwhile, the ovarian germline stem cell niche is built later during late larval stages and requires a two-step process that involves terminal filament formation and cap cell specification. Intriguingly, double security mechanisms of Notch signaling activation coordinated by the soma or the germline control both steps to ensure the robustness of niche assembly. Second, in the vast domains of mammalian cellular signaling, there is an emerging picture of Notch being an active player in a variety of tissues in health and disease. Notch involvement has been shown in stem cell niche establishment in multiple organs, including the brain, muscle, and intestine, where the stem cell niches are essential for the maintenance of adult stem cells. But adult stem cells are not the only cells looking for a home. Cancer stem cells use Notch signaling at specific stages to gain an advantage over endogenous tissue and overpower it, at the same time acquiring migratory and invasive abilities to claim new tissues (e.g., bone) as their territory. Moreover, in vitro models such as organoids reveal similar Notch employment when it comes to the developing stem cell niches. Therefore, a better understanding of the processes regulating stem cell niche assembly is key for the fields of stem cell biology and regenerative medicines.

CRISPR/Cas9 mediated approach to generate YAP-depleted human embryonic stem cell line (MUSIe002-A-1)

Yes-associated protein (YAP), an important effector protein of the Hippo signaling pathway, acts as a molecular switch in controlling cell proliferation and apoptosis. In this study, a YAP-targeted isogenic sub-clone of the MUSIe002-A was generated, designated as MUSIe002-A-1. The MUSIe002-1 cell line had normal pluripotent stem cell characteristics and karyotype. Its ability to differentiate into three germ layers was confirmed. As reduction of YAP does not disturb the pluripotency of hESCs, this cell line serves as a valuable model to extrapolate the functional role of YAP in stem cell biology and its applications.