Stem Cell Physiology Research Articles

Inflammaging, Epigenetic Damage, Mitochondrial Dysfunctions, and Macrophage Alterations are the Main Missing Pieces in the Complex Mosaic of Stem Cell Physiology.

Aging is frequently associated with a progressive increase in chronic low-grade inflammation, known as "inflammaging". Numerous studies have shown that inflammaging is closely linked to the development of several age-related diseases. However, the underlying mechanism and its causal role are still not fully understood despite this association. In the complex context of aging, mesenchymal stem cells (MSCs) undergo changes in behavior and functionality. This narrative topical review examines the recent advances in aging research, specifically focusing on the role of inflammaging and related mechanisms that contribute to age-related chronic diseases. The authors critically investigated whether and how inflammaging, epigenetic damage, mitochondrial changes, and macrophage alterations may influence stem cell behavior, highlighting the interplay between these factors and their potential therapeutic implications. By elucidating the mechanisms underlying these processes, we can gain valuable insights into the maintenance and regeneration of stem cell populations, providing the basis for novel therapeutic strategies targeting age-related decline and disease progression.

"Time Is out of Joint" in Pluripotent Stem Cells: How and Why.

The circadian rhythm is necessary for the homeostasis and health of living organisms. Molecular clocks interconnected by transcription/translation feedback loops exist in most cells of the body. A puzzling exemption to this, otherwise, general biological hallmark is given by the cell physiology of pluripotent stem cells (PSCs) that lack circadian oscillations gradually acquired following their in vivo programmed differentiation. This process can be nicely phenocopied following in vitro commitment and reversed during the reprogramming of somatic cells to induce PSCs. The current understanding of how and why pluripotency is "time-uncoupled" is largely incomplete. A complex picture is emerging where the circadian core clockwork is negatively regulated in PSCs at the post-transcriptional/translational, epigenetic, and other-clock-interaction levels. Moreover, non-canonical functions of circadian core-work components in the balance between pluripotency identity and metabolic-driven cell reprogramming are emerging. This review selects and discusses results of relevant recent investigations providing major insights into this context.

MicroRNAs Regulate Ca2+ Homeostasis in Murine Embryonic Stem Cells.

MicroRNAs (miRNAs) are important regulators of embryonic stem cell (ESC) biology, and their study has identified key regulatory mechanisms. To find novel pathways regulated by miRNAs in ESCs, we undertook a bioinformatics analysis of gene pathways differently expressed in the absence of miRNAs due to the deletion of Dicer, which encodes an RNase that is essential for the synthesis of miRNAs. One pathway that stood out was Ca2+ signaling. Interestingly, we found that Dicer-/- ESCs had no difference in basal cytoplasmic Ca2+ levels but were hyperresponsive when Ca2+ import into the endoplasmic reticulum (ER) was blocked by thapsigargin. Remarkably, the increased Ca2+ response to thapsigargin in ESCs resulted in almost no increase in apoptosis and no differences in stress response pathways, despite the importance of miRNAs in the stress response of other cell types. The increased Ca2+ response in Dicer-/- ESCs was also observed during purinergic receptor activation, demonstrating a physiological role for the miRNA regulation of Ca2+ signaling pathways. In examining the mechanism of increased Ca2+ responsiveness to thapsigargin, neither store-operated Ca2+ entry nor Ca2+ clearance mechanisms from the cytoplasm appeared to be involved. Rather, it appeared to involve an increase in the expression of one isoform of the IP3 receptors (Itpr2). miRNA regulation of Itpr2 expression primarily appeared to be indirect, with transcriptional regulation playing a major role. Therefore, the miRNA regulation of Itpr2 expression offers a unique mechanism to regulate Ca2+ signaling pathways in the physiology of pluripotent stem cells.

Retinoic acid alters metalloproteinase action in red deer antler stem cells.

Metalloproteinases (MMP)s regulate developmental processes, control angiogenesis and wound healing, participate in the formation of immune receptors, and are expressed in stem cells. Retinoic acid (RA) is a potential modulator of these proteinases. The aim was to determine (1) MMPs' action in antler stem cells (ASCs) before and after differentiation into adipo-, osteo-, and chondrocytes and (2) the effect of RA on modifying MMP action in ASCs. Antler tissue from pedicle was collected approximately 40 days after antler casting, post mortem from healthy breeding five year old males (N = 7). The cells were isolated from the pedicle layer of periosteum after skin separation and cultured. The pluripotency of the ASCs was evaluated by mRNA expression for NANOG, SOX2, and OCT4. ASCs were stimulated with RA (100nM) and differentiated for 14 days. The MMP (1-3) and TIMP(1-3) (tissue inhibitor of MMPs) mRNA expression was determined in the ASCs, their concentrations in the ASCs and the medium after RA stimulation as well as profiles of mRNA expression for MMPs: 1-3 and TIMPs: 1-3 during differentiation of ASC to osteocytes, adipocytes and chondrocytes. RA increased MMP-3 and TIMP-3 mRNA expression and output (P < 0.05) and not influenced on MMP-1 and TIMP-1 mRNA expression and output in ASC (P > 0.05). Depending on differentiation of ASC to osteocytes, adipocytes or chondrocytes, MMPs`and TIMPs`expression profile fluctuates for all studied proteases and its inhibitors. The studies demand continuation considering the role of proteases in stem cells physiology and differentiation. The results may be relevant for the study of cellular processes during the cancerogenesis of tumor stem cells.

Artificial induction of circadian rhythm by combining exogenous BMAL1 expression and polycomb repressive complex 2 inhibition in human induced pluripotent stem cells.

Understanding the physiology of human-induced pluripotent stem cells (iPSCs) is necessary for directed differentiation, mimicking embryonic development, and regenerative medicine applications. Pluripotent stem cells (PSCs) exhibit unique abilities such as self-renewal and pluripotency, but they lack some functions that are associated with normal somatic cells. One such function is the circadian oscillation of clock genes; however, whether or not PSCs demonstrate this capability remains unclear. In this study, the reason why circadian rhythm does not oscillate in human iPSCs was examined. This phenomenon may be due to the transcriptional repression of clock genes resulting from the hypermethylation of histone H3 at lysine 27 (H3K27), or it may be due to the low levels of brain and muscle ARNT-like 1 (BMAL1) protein. Therefore, BMAL1-overexpressing cells were generated and pre-treated with GSK126, an inhibitor of enhancer of zest homologue 2 (EZH2), which is a methyltransferase of H3K27 and a component of polycomb repressive complex 2. Consequently, a significant circadian rhythm following endogenous BMAL1, period 2 (PER2), and other clock gene expression was induced by these two factors, suggesting a candidate mechanism for the lack of rhythmicity of clock gene expression in iPSCs.

Loss of LRP1 in Adult Neural Stem Cells Impairs Migration to Ischemic Lesions.

After ischemia, cells in the brain parenchyma upregulate stromal derived factor 1 (SDF1), driving chemokine receptor CXCR4-mediated migration of adult neural stem cells to the ischemic injury. We discovered a novel regulator of CXCR4 in neural stem cells, low-density lipoprotein receptor related protein 1 (LRP1). We used Nestin-driven knockout of LRP1 and induction of td-tomato in neural stem cells of adult mice. We observed reduced localization of td-tomato positive cells to the lesion, and find disrupted CXCR4-mediated neural stem cell migration in vitro, which is likely driven by LRP1-mediated loss of CXCR4 expression in vivo. Our results suggest that LRP1 is a novel regulator of CXCR4 in neural stem cells. This heretofore unknown interaction between LRP1 and CXCR4 could have significant consequences for multiple aspects of neural stem cell physiology.

Uncontrolled neural stem cell proliferation affects larval survival during dietary nutrient withdrawal.

Regulated stem cell proliferation during development is critical to produce and organize all the cell types required in the adult organism. In some developing organisms that experience intermittent periods of starvation, stem cell physiology adapts dynamically, presumably to ensure optimal development. In the developing Drosophila brain, there are two populations of neuroblasts (neural stem cells) that respond differently to dietary nutrient starvation: one subtype is sensitive to systemic nutrient levels and ceases dividing, while the other continues to divide regardless of dietary nutrient availability. Previous work has shown that this behavior is mediated by a cell-intrinsic genetic program involving the Eyeless and myc transcription factors. We set out to understand whether arrest of neuroblast proliferation plays a role in larval survival during starvation. We forced all neuroblasts in the developing brain to continue to proliferate during dietary nutrient withdrawal by overexpressing myc and Eyeless and measured larval survival over time. Larvae with abnormal enforced neuroblast proliferation died at a faster rate than control larvae, suggesting that the arrest of neuroblast proliferation is critical for larval survival during periods of starvation. Our results are consistent with the idea that neuroblasts which arrest their proliferation are involved in reallocating nutrients to the other persistently dividing neuroblast subtype in a form of nervous system sparing.

Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP.

ESTABLISHMENT AND TREATMENT OF MURINE COLONOIDS TO STUDY THE EPITHELIAL EFFECTS OF INTESTINAL INFLAMMATION IN VITRO

Disruption of the intestinal epithelial barrier is a hallmark of inflammatory bowel disease (IBD). The 3-dimensional colonoid culture system is a useful in vitro model to study intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice undergoing acute inflammation using a DSS model and primarily reflect stem cell physiology rather than that of terminally differentiated cells. We developed experimental methods to address this limitation. We terminally differentiate colonoids or create 2-D monolayers, and challenge them with a cocktail of inflammatory mediators that are typically elevated during IBD.

ESTABLISHMENT AND TREATMENT OF MURINE COLONOIDS TO STUDY THE EPITHELIAL EFFECTS OF INTESTINAL INFLAMMATION IN VITRO

Abstract BACKGROUND Disruption of the intestinal epithelial barrier is a hallmark of inflammatory bowel disease (IBD). The 3-dimensional colonoid culture system is a useful in vitro model to study intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice undergoing acute inflammation using a DSS model and primarily reflect stem cell physiology rather than that of terminally differentiated cells. We developed experimental methods to address this limitation. We terminally differentiate colonoids or create 2-D monolayers, and challenge them with a cocktail of inflammatory mediators that are typically elevated during IBD. METHODS Murine colonoids are established by isolating colonic crypts and plating them in basement membrane matrix. Over 3 to 5 days, crypt structures develop into spheroid colonoids 250 μm in diameter, forming a prominent lumen surrounded by a thin layer of epithelial stem cells. They are passaged a minimum of two times and then are either 1) terminally differentiated by incubating the colonoids with Differentiating Media or 2) used to establish intestinal epithelial monolayers. Inflammation is then induced by incubating cells with a cocktail of inflammatory mediators (TNFa, IL-1b, IFNg, H2O2). Protein is collected after 72 hours, and expression of proteins involved in mitochondrial biogenesis were assessed (n=3). RESULTS We found that changes in mitochondrial dynamics, reflective of key metabolic changes in the epithelium that are observed in colonic scrapings of DSS-treated mice are not recapitulated in traditional colonoids derived from mice on the same DSS regimen. We found that changes in expression and protein levels of peroxisome proliferator-activator receptor-gamma coactivator-1a (PGC1a), the master regulator protein of mitochondrial biogenesis, were not maintained in colonoids. After terminally differentiating colonoids and subsequently inducing inflammation, we were able to recapitulate a failure of mitochondrial biogenesis failure that is a hallmark of both human IBD and murine colitis. Protein expression of PGC1a as well as downstream transcription factor A, mitochondria (TFAM) were both decreased when assessed via western blot. DISCUSSION Colonoids are a useful tool for studying colonic physiology in vitro, however are limited in their ability to evaluate the effects of in vivo inflammation on the mitochondrial function and cellular metabolism. When removed from the intestinal milieu, crypt cells demonstrate a recovery of mitochondrial health and reflect stem primarily stem cell physiology. Our strategies provide opportunities for organoid manipulation which, when employed properly, can shed light on key aspects of intestinal epithelial mitochondrial function during inflammation.

Modeling Premalignant Transformation of Hematopoietic Stem Cells in a Nanobioreactor in Microgravity

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twin study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation, such as increased chromosome translocations and inversions, occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA's Countermeasures Program found significant similarities between the multisystem physiological toxicities experienced in cancer patients as well as during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system's ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduced a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we are analyzing cytokine profiles and mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). The first batch of bioreactors launched with the SpX-24 mission in December 2021 and the second batch is currently on orbit as part of the SpX-25 mission. This investigation has the potential to provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, HSC response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

Abstract 3154: Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity

Abstract Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twins study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation such as increased chromosome translocations and inversions occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA’s Countermeasures Program found significant similarities between the multisystem physiological toxicities in cancer patients and during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system’s ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduce a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we will be analyzing mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). Our bioreactors are scheduled to launch as part of the SpaceX CRS-24 mission on Dec 21, 2021. This investigation may provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells. Citation Format: Luisa Ladel, Jessica Pham, Isabelle Oliver, Larisa Balaian, Catriona HM Jamieson. Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3154.

Self-Sustained Regulation or Self-Perpetuating Dysregulation: ROS-dependent HIF-YAP-Notch Signaling as a Double-Edged Sword on Stem Cell Physiology and Tumorigenesis.

Organ development, homeostasis, and repair often rely on bidirectional, self-organized cell-niche interactions, through which cells select cell fate, such as stem cell self-renewal and differentiation. The niche contains multiplexed chemical and mechanical factors. How cells interpret niche structural information such as the 3D topology of organs and integrate with multiplexed mechano-chemical signals is an open and active research field. Among all the niche factors, reactive oxygen species (ROS) have recently gained growing interest. Once considered harmful, ROS are now recognized as an important niche factor in the regulation of tissue mechanics and topology through, for example, the HIF-YAP-Notch signaling pathways. These pathways are not only involved in the regulation of stem cell physiology but also associated with inflammation, neurological disorder, aging, tumorigenesis, and the regulation of the immune checkpoint molecule PD-L1. Positive feedback circuits have been identified in the interplay of ROS and HIF-YAP-Notch signaling, leading to the possibility that under aberrant conditions, self-organized, ROS-dependent physiological regulations can be switched to self-perpetuating dysregulation, making ROS a double-edged sword at the interface of stem cell physiology and tumorigenesis. In this review, we discuss the recent findings on how ROS and tissue mechanics affect YAP-HIF-Notch-PD-L1 signaling, hoping that the knowledge can be used to design strategies for stem cell-based and ROS-targeting therapy and tissue engineering.

Stem cell-based therapies for musculoskeletal regeneration: Role of the microenvironment in augmenting regenerative medicine therapies by elucidating stem cell physiology

Stem cell-based therapies for musculoskeletal regeneration: Role of the microenvironment in augmenting regenerative medicine therapies by elucidating stem cell physiology

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.

Ret kinase-mediated mechanical induction of colon stem cells by tumor growth pressure stimulates cancer progression in vivo

How mechanical stress actively impacts the physiology and pathophysiology of cells and tissues is little investigated in vivo. The colon is constantly submitted to multi-frequency spontaneous pulsatile mechanical waves, which highest frequency functions, of 2 s period, remain poorly understood. Here we find in vivo that high frequency pulsatile mechanical stresses maintain the physiological level of mice colon stem cells (SC) through the mechanosensitive Ret kinase. When permanently stimulated by a magnetic mimicking-tumor growth analogue pressure, we find that SC levels pathologically increase and undergo mechanically induced hyperproliferation and tumorigenic transformation. To mimic the high frequency pulsatile mechanical waves, we used a generator of pulsed magnetic force stimulation in colonic tissues pre-magnetized with ultra-magnetic liposomes. We observed the pulsatile stresses using last generation ultra-wave dynamical high-resolution imaging. Finally, we find that the specific pharmacological inhibition of Ret mechanical activation induces the regression of spontaneous formation of SC, of CSC markers, and of spontaneous sporadic tumorigenesis in Apc mutated mice colons. Consistently, in human colon cancer tissues, Ret activation in epithelial cells increases with tumor grade, and partially decreases in leaking invasive carcinoma. High frequency pulsatile physiological mechanical stresses thus constitute a new niche that Ret-dependently fuels mice colon physiological SC level. This process is pathologically over-activated in the presence of permanent pressure due to the growth of tumors initiated by pre-existing genetic alteration, leading to mechanotransductive self-enhanced tumor progression in vivo, and repressed by pharmacological inhibition of Ret.

Promyelocytic leukemia protein (PML) and stem cells: from cancer to pluripotency

The promyelocytic leukemia protein (PML) is the core organizer of cognate nuclear bodies (PML-NBs). Through physical interaction or modification of diverse protein clients, PML-NBs regulate a multitude of - often antithetical- biological processes such as antiviral and stress response, inhibition of cell proliferation and autophagy, and promotion of apoptosis or senescence. Although PML was originally recognized as a tumor-suppressive factor, more recent studies have revealed a "double-faced" agent role for PML. Indeed, PML displayed tumor cell pro-survival and pro-migratory functions via inhibition of migration suppressing molecules or promotion of transforming growth factor beta (TGF-β) mediated Epithelial-Mesenchymal Transition (EMT) that may promote cancer cell dissemination. In this line, PML was found to correlate with poor patient prognosis in distinct tumor contexts. Furthermore, in the last decade, a number of publications have implicated PML in the physiology of normal or cancer stem cells (CSCs). Promyelocytic leukemia protein activates fatty acid oxidation (FAO), a metabolic mechanism required for the asymmetric divisions and maintenance of hematopoietic stem cells (HSCs). In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PML is required for maintenance of the naïve and acquisition of the induced pluripotency state, respectively. Correspondingly, PML ablation causes significant morphological gene expression and lineage choice changes. In this review, we focus on the mechanisms orchestrated by PML and PML-NBs in cancer and healthy stem cells, from cell physiology to the regulation of chromatin dynamics.

MTHFR C677T mutation affects adipogenic differentiation abilities of human bone marrow-derived mesenchymal stem cells.

The effects of 5,10-Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) viability, morphology, physiology and differentiation capacity were investigated in this study. For this purpose, primary hBM-MSCs with wild type (WT, C/C), heterozygote (HTZ, C/T) and homozygote (HMZ, T/T) for the MTHFR gene were obtained with ethical committee permission and donor informed. Mutations were detected using RFLP and Sanger sequencing methods from genomic DNA isolated from cells, colonization properties were investigated by CFU-F test and proliferative differences were investigated by MTT test. Adipogenic, osteogenic, and chondrogenic differentiation were induced to study changes in their differentiation potentials, and the results were statistically analyzed using one-way ANOVA with Graphpad Prism. A total of 13 donors were screened and there were no differences in the hBM-MSC markers and in vitro morphologies of the cells. While there were significant differences between WT and HTZ as a result of the CFU-F test, there were no significant differences in the MTT test after 24 and 48 h. As a result of differentiation tests, it was found that adipogenic differentiation was significantly more in HMZ cells than WT cells. Osteogenic and chondrogenic differentiation results did not give statistically significant results. As a result of these experiments, adipogenic differentiation was found to be affected by the MTHFR genotype in hBM-MSCs.

Plexin-B2 orchestrates collective stem cell dynamics via actomyosin contractility, cytoskeletal tension and adhesion

During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thus establish a significant function of Plexin-B2 in orchestrating cytoskeletal tension and cell-cell/cell-matrix adhesion, therefore solidifying the importance of collective cell mechanics in governing stem cell physiology and tissue morphogenesis.

Domestication of chemicals attacking metazoan embryogenesis: identification of safe natural products modifying developmental signaling pathways in human.

Soil microorganisms are rich sources of bioactive natural products. Interspecies interactions are the cues of their production and refine biological activities. These interactions in natural environments include the interplay between microorganisms and Metazoans (animals), such as nematodes, insects, and ticks. Chemical intercellular communication modulators could exert ideal Metazoan-selective toxicity for defending microorganisms. Developmental signaling pathways, such as the Notch, TGF-beta, and Wnt pathways, are intercellular communication networks that contribute to the reproducible formation of complex higher-order Metazoan body structures. Natural modifiers of the developmental signaling pathway are attractive therapeutic seeds for carcinoma and sarcoma treatment. However, these fundamental signaling pathways also play indispensable physiological roles and their perturbation could lead to toxicity, such as defects in stem cell physiology and tissue regeneration processes. In this review, we introduce a screening system that selects developmental signaling inhibitors with wide therapeutic windows using zebrafish embryonic phenotypes and provide examples of microorganism-derived Wnt pathway inhibitors. Moreover, we discuss safety prospects of the developmental signaling inhibitors.