Controlling human organoid symmetry breaking reveals signaling gradients drive segmentation clock waves.

Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.

Human Fetal TNF-α-Cytokine-Producing CD4+ Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life.

Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)+CD4+CD69+ T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4+ T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4+ T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4+ Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4+ T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.

Axial elongation of caudalized human organoids mimics aspects of neural tube development.

Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development. Elongating organoids consist largely of neuroepithelial compartments and contain TBXT+SOX2+ neuro-mesodermal progenitors in addition to PAX6+NES+ neural progenitors. A critical threshold of Wnt agonism stimulated singular axial extensions while maintaining multiple cell lineages, such that organoids displayed regionalized anterior-to-posterior HOX gene expression with hindbrain (HOXB1) regions spatially distinct from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR interference-mediated silencing of TBXT, a Wnt pathway target, increased neuroepithelial compartmentalization, abrogated HOX expression and disrupted uniaxial elongation. Together, these results demonstrate the potent capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early human nervous system development.

Computational single-cell methods for predicting cancer risk.

Despite recent biotechnological breakthroughs, cancer risk prediction remains a formidable computational and experimental challenge. Addressing it is critical in order to improve prevention, early detection and survival rates. Here, I briefly summarize some key emerging theoretical and computational challenges as well as recent computational advances that promise to help realize the goals of cancer-risk prediction. The focus is on computational strategies based on single-cell data, in particular on bottom-up network modeling approaches that aim to estimate cancer stemness and dedifferentiation at single-cell resolution from a systems-biological perspective. I will describe two promising methods, a tissue and cell-lineage independent one based on the concept of diffusion network entropy, and a tissue and cell-lineage specific one that uses transcription factor regulons. Application of these tools to single-cell and single-nucleus RNA-seq data from stages prior to invasive cancer reveal that they can successfully delineate the heterogeneous inter-cellular cancer-risk landscape, identifying those cells that are more likely to turn cancerous. Bottom-up systems biological modeling of single-cell omic data is a novel computational analysis paradigm that promises to facilitate the development of preventive, early detection and cancer-risk prediction strategies.

Human amniotic membrane scaffold enhances adipose mesenchymal stromal cell mitochondrial bioenergetics promoting their regenerative capacities.

The human amniotic membrane (hAM) has been applied as a scaffold in tissue engineering to sustain stem cells and enhance their regenerative capacities. We investigated the molecular and biochemical regulations of mesenchymal stromal cells (MSCs) cultured on hAM scaffold in a three-dimensional (3D) setting. Culture of adipose-MSCs (AMSCs) on decellularized hAM showed significant improvement in their viability, proliferative capacity, resistance to apoptosis, and enhanced MSC markers expression. These cultured MSCs displayed altered expression of markers associated with pro-angiogenesis and inflammation and demonstrated increased potential for differentiation into adipogenic and osteogenic lineages. The hAM scaffold modulated cellular respiration by upregulating glycolysis in MSCs as evidenced by increased glucose consumption, cellular pyruvate and lactate production, and upregulation of glycolysis markers. These metabolic changes modulated mitochondrial oxidative phosphorylation (OXPHOS) and altered the production of reactive oxygen species (ROS), expression of OXPHOS markers, and total antioxidant capacity. They also significantly boosted the urea cycle and altered the mitochondrial ultrastructure. Similar findings were observed in bone marrow-derived MSCs (BMSCs). Live cell imaging of BMSCs cultured in the same 3D environment revealed dynamic changes in cellular activity and interactions with its niche. These findings provide evidence for the favorable properties of hAM as a biomimetic scaffold for enhancing the in vitro functionality of MSCs and supporting their potential usefulness in clinical applications.

ATF6 is essential for human cone photoreceptor development.

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

Cell and Tissue Therapy for the Treatment of Chronic Liver Disease.

Liver disease is an important clinical problem, impacting 600 million people worldwide. It is the 11th-leading cause of death in the world. Despite constant improvement in treatment and diagnostics, the aging population and accumulated risk factors led to increased morbidity due to nonalcoholic fatty liver disease and steatohepatitis. Liver transplantation, first established in the 1960s, is the second-most-common solid organ transplantation and is the gold standard for the treatment of liver failure. However, less than 10% of the global need for liver transplantation is met at the current rates of transplantation due to the paucity of available organs. Cell- and tissue-based therapies present an alternative to organ transplantation. This review surveys the approaches and tools that have been developed, discusses the distinctive challenges that exist for cell- and tissue-based therapies, and examines the future directions of regenerative therapies for the treatment of liver disease.

The Hair Follicle as an Interdisciplinary Model for Biomedical Research: An Eclectic Literature Synthesis.

Skin is a comparatively accessible organ possessing many conserved regulatory and signaling pathways, drawing researchers from varied fields toward its study. Hair follicle (HF) biology in particular has expanded rapidly over the preceding decade, helping to shape and develop scientific knowledge across diverse areas of biomedical research, beyond the skin. The hope in compiling this review is to inspire more researchers to utilize the HF as an instructive biological model, bringing with them fresh perspectives and experience from differing fields of study. The authors also wish to further motivate seasoned hair researchers to explore the further reaches of their understanding and the discoveries yet to be made. For this reason, the authors have endeavored to collate an eclectic mix of some of the most thought-provoking and scientifically intriguing articles associated with the field of HF research, published in the preceding two years.

Progress in Bioengineering Strategies for Heart Regenerative Medicine.

The human heart has the least regenerative capabilities among tissues and organs, and heart disease continues to be a leading cause of mortality in the industrialized world with insufficient therapeutic options and poor prognosis. Therefore, developing new therapeutic strategies for heart regeneration is a major goal in modern cardiac biology and medicine. Recent advances in stem cell biology and biotechnologies such as human pluripotent stem cells (hPSCs) and cardiac tissue engineering hold great promise for opening novel paths to heart regeneration and repair for heart disease, although these areas are still in their infancy. In this review, we summarize and discuss the recent progress in cardiac tissue engineering strategies, highlighting stem cell engineering and cardiomyocyte maturation, development of novel functional biomaterials and biofabrication tools, and their therapeutic applications involving drug discovery, disease modeling, and regenerative medicine for heart disease.

Generative Adversarial Networks for Morphological-Temporal Classification of Stem Cell Images.

Frequently, neural network training involving biological images suffers from a lack of data, resulting in inefficient network learning. This issue stems from limitations in terms of time, resources, and difficulty in cellular experimentation and data collection. For example, when performing experimental analysis, it may be necessary for the researcher to use most of their data for testing, as opposed to model training. Therefore, the goal of this paper is to perform dataset augmentation using generative adversarial networks (GAN) to increase the classification accuracy of deep convolutional neural networks (CNN) trained on induced pluripotent stem cell microscopy images. The main challenges are: 1. modeling complex data using GAN and 2. training neural networks on augmented datasets that contain generated data. To address these challenges, a temporally constrained, hierarchical classification scheme that exploits domain knowledge is employed for model learning. First, image patches of cell colonies from gray-scale microscopy images are generated using GAN, and then these images are added to the real dataset and used to address class imbalances at multiple stages of training. Overall, a 2% increase in both true positive rate and F1-score is observed using this method as compared to a straightforward, imbalanced classification network, with some greater improvements on a classwise basis. This work demonstrates that synergistic model design involving domain knowledge is key for biological image analysis and improves model learning in high-throughput scenarios.

In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells.

During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440.

Microenvironmental Regulation of Long Noncoding RNA LINC01133 Promotes Cancer Stem Cell-Like Phenotypic Traits in Triple-Negative Breast Cancers.

The fibrotic tumor microenvironment is a critical player in the pathogenesis of triple-negative breast cancers (TNBCs), with the presence of fibroblastic infiltrates particularly correlating with tumors that are clinically advanced. On this front, we previously demonstrated that TNBCs are highly enriched in fibroblastic stromal progenitor cells called mesenchymal stem/stromal cells (MSCs) and that such cells play critical roles in promoting TNBC initiation and progression. How TNBC cells respond to MSC stimulation, however, is not fully understood, and stands to reveal contextual signals used by TNBC cells during tumor development and provide biomarkers and therapeutic targets of pertinence to TNBC management. Here, we report that MSCs strongly induced the long noncoding RNA (lncRNA) LINC01133 in neighboring TNBC cells. Indeed, although lncRNAs have been tightly associated with cancer development, their contributions to breast cancer in general, and to TNBC pathogenesis in particular, have not been fully elucidated, and we set out to determine if LINC01133 regulated malignant traits in TNBC cells. We establish that LINC01133 is sufficient, on its own, in promoting phenotypic and growth characteristics of cancer stem cell-like cells, and that it is a direct mediator of the MSC-triggered miR-199a-FOXP2 pathway in TNBC models. Furthermore, we show that LINC01133 is a critical regulator of the pluripotency-determining gene Kruppel-Like Factor 4 (KLF4), and that it represents a biomarker and prognosticator of disease outcome in the clinic. Collectively, our findings introduce LINC01133 as a novel functional driver of malignancy and a potential theranostic in TNBC. Stem Cells 2019;37:1281-1292.

Impact of CD34+ cell dose on reduced intensity conditioning regimen haploidentical hematopoietic stem cell transplantation.

OBJECTIVES: Haploidentical hematopoietic stem cell transplant (haplo-SCT) has been associated with higher rates of graft rejection, and a higher dose of CD34+ cell dose is frequently requested. We aim to explore the impact of CD34+ cell dose in peripheral blood stem cell (PBSC) grafts using reduced intensity conditioning (RIC) in haplo-SCT. METHODS: Sixty-eight consecutive haplo-SCT in adult patients were included. Graft-vs-host disease (GVHD) prophylaxis consisted on ATG, PTCy, and CsA. The cohort was divided in two groups using CD34+ dose of ≥ 9 × 106 CD34+/Kg as cutoff point. Median follow-up was 8.9 months. RESULTS: Median cell dose infused was 9.32 × 106 CD34+/Kg. Forty (58.8%) recipients received grafts with CD34+ cells ≥9 × 106 /kg. The infusion ≥ 9 × 106 CD34+/Kg cell dose had a negative impact in overall survival (P = .03) after adjusting for age at transplant. The cumulative incidence of acute GVHD and graft failure were not significantly influenced per CD34+ cell dose. Only four recipients had grade III aGVHD, and all of them received grafts with a CD34+ cell dose ≥ 9 × 106 . CONCLUSION: In RIC haplo-SCT, recipients may not benefit from PBSC grafts with a CD34+/kg cell dose higher than 9 × 106 cells/kg, as it can have an adverse impact in post-transplant outcome.

Human embryonic stem cells: Distinct molecular personalities and applications in regenerative medicine.

The field of stem cell biology is exciting because it provides researchers and clinicians with seemingly unlimited applications for treating many human diseases. Stem cells are a renewable source of pluripotent cells that can differentiate into nearly all human cell types. In this article we focus particularly on human embryonic stem (hES) cells, derived from the inner cell mass of the blastocyst and cultured for expansion while remaining undifferentiated, to explore their unique molecular personalities and clinical applications. The aim of this literature review is to reflect the interest in hES cells and to provide a resource for researchers and clinicians interested in the molecular characteristics of such cells. Clin. Anat. 32:354-360, 2019. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Concise Review: An (Im)Penetrable Shield: How the Tumor Microenvironment Protects Cancer Stem Cells.

Cancer stem cells (CSCs) are defined by their unlimited self-renewal ability and their capacity to initiate and maintain malignancy, traits that are not found in most cells that comprise the tumor. Although current cancer treatments successfully reduce tumor burden, the tumor will likely recur unless CSCs are effectively eradicated. This challenge is made greater by the protective impact of the tumor microenvironment (TME), consisting of infiltrating immune cells, endothelial cells, extracellular matrix, and signaling molecules. The TME acts as a therapeutic barrier through immunosuppressive, and thereby tumor-promoting, actions. These factors, outside of the cancer cell lineage, work in concert to shelter CSCs from both the body's intrinsic anticancer immunity and pharmaceutical interventions to maintain cancer growth. Emerging therapies aimed at the TME offer a promising new tool in breaking through this shield to target the CSCs, yet definitive treatments remain unrealized. In this review, we summarize the mechanisms by which CSCs are protected by the TME and current efforts to overcome these barriers. Stem Cells 2017;35:1123-1130.

Second-line treatment for acute graft-versus-host disease with mesenchymal stromal cells: A decision model.

OBJECTIVE: No standard second-line treatment exists for acute graft-versus-host disease steroid-refractory (SR-aGvHD), and long-term outcomes remain poor. Mesenchymal stromal cells (MSCs) have been evaluated as treatment, but no disease model (DM) exists that integrates and extrapolates currently available evidence. The aim of this study was to develop such a DM to describe the natural history of SR-aGvHD and to predict long-term outcomes. METHOD: The DM was developed in collaboration with experts in haematology-oncology. Subsequently, a model simulation was run. Input parameters for transition and survival estimates were informed by published data of clinical trials on MSC treatment for SR-aGvHD. Parametric distributions were used to estimate long-term survival rates after MSCs. RESULTS: The newly developed DM is a cohort model that consists of eight health states. For the model simulation, we obtained data on 327 patients from 14 published phase II trials. Due to limited evidence, DM structure was simplified and several assumptions had to be made. Median overall survival was 3.2 years for complete response and 0.5 years for no complete response. CONCLUSION: The DM provides a comprehensive overview on the second-line treatment pathway for aGvHD and enables long-term predictions that can be used to perform a cost-effectiveness analysis comparing any treatment for SR-aGvHD.

Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.

Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.

Entropy Density Acceleration and Minimum Dissipation Principle: Correlation with Heat and Matter Transfer in Glucose Catabolism.

The heat and matter transfer during glucose catabolism in living systems and their relation with entropy production are a challenging subject of the classical thermodynamics applied to biology. In this respect, an analogy between mechanics and thermodynamics has been performed via the definition of the entropy density acceleration expressed by the time derivative of the rate of entropy density and related to heat and matter transfer in minimum living systems. Cells are regarded as open thermodynamic systems that exchange heat and matter resulting from irreversible processes with the intercellular environment. Prigogine's minimum energy dissipation principle is reformulated using the notion of entropy density acceleration applied to glucose catabolism. It is shown that, for out-of-equilibrium states, the calculated entropy density acceleration for a single cell is finite and negative and approaches as a function of time a zero value at global thermodynamic equilibrium for heat and matter transfer independently of the cell type and the metabolic pathway. These results could be important for a deeper understanding of entropy generation and its correlation with heat transfer in cell biology with special regard to glucose catabolism representing the prototype of irreversible reactions and a crucial metabolic pathway in stem cells and cancer stem cells.

Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells.

Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2(-/-) NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321-1331.

Concomitant MDS with isolated 5q deletion and MGUS: case report and review of molecular aspects.

Patients with monoclonal gammopathy of undetermined significance (MGUS) have a higher risk for the development of concomitant primary cancers such as multiple myeloma (MM) and myelodysplastic syndrome (MDS). We report the case of patient initially suffering from MGUS of the IgG lambda subtype for more than 10 yr, which evolved to MM and MDS with deletion (5q) with severe pancytopenia. Due to pancytopenia, he received dose-reduced treatment with lenalidomide and dexamethasone. He achieved an ongoing transfusion independency after about 1 month of treatment. Bone marrow taken 14 months after start of treatment showed a complete cytogenetic response of the del(5q) clone and a plasma cell infiltration below 5%. In contrast to the development of MM in MGUS patients, the subsequent occurrence of MDS after diagnosis of MGUS is infrequent. Moreover, the biological association of MDS with MGUS is not sufficiently understood, but the non-treatment-related occurrence supports the pathogenetic role of pre-existing alterations of stem cells. Here, we summarize data on concomitant MDS and MGUS/MM with particular emphasis on molecular aspects.