Constricted migration modulates stem cell differentiation

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis increases—which is relevant to bone and to calcified fibrotic tissues, including diseased muscle. Tissue repair can thus be modulated up or down by the curvature of pores through which stem cells squeeze.

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STEM-20. RADIO-SENSITIZING GLIOBLASTOMA CANCER STEM CELLS BY INHIBITION OF FACT

Neuro-Oncology ◽

10.1093/neuonc/noz175.993 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi237-vi238

Author(s):

Miranda Montgomery ◽

Abigail Zalenski ◽

Amanda Deighen ◽

Sherry Mortach ◽

Treg Grubb ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Cancer Stem Cells ◽

Combination Therapy ◽

High Rate ◽

Primary Role ◽

Cancer Cell Growth ◽

Treatment Paradigm

Abstract Glioblastoma (GBM) has a particularly high rate of recurrence with a 5-year overall survival rate of approximately 5%. This is in part due to a sub-population of cancer stem cells (CSC), which are both radioresistant and chemotherapeutically resistant to conventional treatments. Here we investigated CBL0137, a small molecule form of curaxin, in combination with radiotherapy as a means to radiosensitize CSCs. CBL0137 sequesters FACT (facilitates chromatin transcription) complex to chromatin, which leads to activation of p53 and inhibition of NF-κB. This sequestering of FACT results in cytotoxicity especially within tumor cells and prevents FACT from performing its primary role as a histone chaperone, as well as inhibits its part in the DNA damage response pathway. We show that when combined with radiotherapy, CBL0137 administration limited the ability of CSCs to identify and repair damaged DNA. CSCs treated in vitro with CBL0137 and irradiation showed an increased inhibition of cancer cell growth and decreased viability compared to irradiation or drug alone. Combination therapy also showed more DNA damage in the CSCs than with either agent alone. Based on our in vitro evidence for the efficacy of combination therapy to target CSCs, we moved forward to test the treatment in vivo. Using a subcutaneous model, we show that the amount of CD133+ cells (a marker for GMB CSCs) was reduced in irradiation plus CBL0137 compared to either treatment alone. Survival studies demonstrated that irradiation plus CBL0137 compared to irradiation alone or CBL0137 alone increase lifespan. Here we show the ability of CBL0137, in combination with irradiation, to target patient GBM CSCs both in vitro and in vivo. This work establishes a new treatment paradigm for GBM that inclusively targets CSCs and may ultimately reduce tumor recurrence.

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Time-Dependent Reduction of Calcium Oscillations in Adipose-Derived Stem Cells Differentiating towards Adipogenic and Osteogenic Lineage

Biomolecules ◽

10.3390/biom11101400 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1400

Author(s):

Enrico C. Torre ◽

Mesude Bicer ◽

Graeme S. Cottrell ◽

Darius Widera ◽

Francesco Tamagnini

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Adipogenic Differentiation ◽

Specific Treatment ◽

Stem Cell Differentiation ◽

Calcium Oscillations ◽

Multipotent Stem Cells ◽

Over Time

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.

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CBIO-13. THOC1 DRIVES GBM AGGRESSION THROUGH MODULATION OF R-LOOPS AND GENOMIC STABILITY

Neuro-Oncology ◽

10.1093/neuonc/noab196.113 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi29-vi30

Author(s):

Shreya Budhiraja ◽

Shivani Baisiwala ◽

Khizar Nandoliya ◽

Li Chen ◽

Crismita Dmello ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Neural Stem Cells ◽

Histone Deacetylase ◽

Genomic Stability ◽

Loop Formation ◽

Driver Genes ◽

A Genome

Abstract Glioblastoma (GBM) is the most aggressive and common type of adult malignant brain tumor, with a median survival of only 21 months. To identify which genes drive its highly aggressive phenotype, we performed a genome-wide CRISPR-Cas9 knockout screen. Results showed substantial enrichment of ~160 novel essential oncogenic driver genes and pathways, including a previously unstudied gene THOC1—involved in RNA processing—that showed significant elevations in expression at RNA and protein levels (p< 0.05) in GBM, as well as a significant survival benefit in patient datasets when downregulated (p< 0.05). Knocking out THOC1 resulted in cell death in multiple GBM patient-derived xenograft (PDX) lines and extended survival compared to the controls (p< 0.01) in vivo. Overexpression of THOC1 in neural stem cells resulted in transformation to a cancerous phenotype, as evidenced by sphere formation in a soft agar assay (p< 0.01) and in vivo tumor engraftment assays. Further investigation of THOC1 through immunoprecipitation in neural stem cells and multiple GBM lines showed significant interaction in GBM with histone deacetylase complex SIN3A, involved in recruiting major histone deacetylases in order to close the DNA and prevent the accumulation of R-loops, RNA:DNA hybrids that pose a threat to genomic stability. Additional investigation revealed that THOC1-knockdowns in vitro induced R-loop formation and DNA damage, while THOC1-overexpression in vitro resulted in an untenable decrease in R-loops and DNA damage, suggesting that the THOC1-SIN3A axis is elevated in GBM in order to prevent the accumulation of genotoxic R-loops. Additionally, histone deacetylase activity was shown to be elevated in THOC1-overexpression conditions and reduced in THOC1-knockdown conditions, confirming that the THOC1-SIN3A axis functions to prevent R-loop accumulation through the epigenetic regulation. In summary, our whole-genome CRISPR-Cas9 knockout screen has identified a promising therapeutic target for GBM—a disease desperately in need of therapeutic innovations.

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Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

Stem Cells International ◽

10.1155/2016/5481493 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 38

Author(s):

Angela Maria Cozzolino ◽

Valeria Noce ◽

Cecilia Battistelli ◽

Alessandra Marchetti ◽

Germana Grassi ◽

...

Keyword(s):

Stem Cells ◽

Culture Method ◽

Cell Types ◽

Culture Conditions ◽

Precursor Cells ◽

Substrate Stiffness ◽

Growth And Survival ◽

Liver Stem Cells

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that,in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines asin vitromodels of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

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Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

Biomolecules ◽

10.3390/biom10091306 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1306

Author(s):

Ann-Kristin Afflerbach ◽

Mark D. Kiri ◽

Tahir Detinis ◽

Ben M. Maoz

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Types ◽

In Vitro Models ◽

Cell Source ◽

Fundamental Research ◽

Multiple Cell ◽

Vitro Model

The human-relevance of an in vitro model is dependent on two main factors—(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

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Mesenchymal stem cells are injured by complement after their contact with serum

Blood ◽

10.1182/blood-2012-03-420612 ◽

2012 ◽

Vol 120 (17) ◽

pp. 3436-3443 ◽

Cited By ~ 94

Author(s):

Yan Li ◽

Feng Lin

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Surface ◽

Complement Activation ◽

Human Mscs ◽

Cellular Injury ◽

Allogeneic Mscs ◽

Complement Regulators

Abstract Despite the potent immunosuppressive activity that mesenchymal stem cells (MSCs) display in vitro, recent clinical trial results are disappointing, suggesting that MSC viability and/or function are greatly reduced after infusion. In this report, we demonstrated that human MSCs activated complement of the innate immunity after their contact with serum. Although all 3 known intrinsic cell-surface complement regulators were present on MSCs, activated complement overwhelmed the protection of these regulators and resulted in MSCs cytotoxicity and dysfunction. In addition, autologous MSCs suffered less cellular injury than allogeneic MSCs after contacting serum. All 3 complement activation pathways were involved in generating the membrane attack complex to directly injure MSCs. Supplementing an exogenous complement inhibitor, or up-regulating MSC expression levels of CD55, one of the cell-surface complement regulators, helped to reduce the serum-induced MSC cytotoxicity. Finally, adoptively transferred MSCs in complement deficient mice or complement-depleted mice showed reduced cellular injury in vivo compared with those in wild type mice. These results indicate that complement is integrally involved in recognizing and injuring MSCs after their infusion, suggesting that autologous MSCs may have ad-vantages over allogeneic MSCs, and that inhibiting complement activation could be a novel strategy to improve existing MSC-based therapies.

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Distribution of Seeded Mesenchymal Stem Cells on Hydroxyapatite Porous Ceramics

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.330-332.1141 ◽

2007 ◽

Vol 330-332 ◽

pp. 1141-1144 ◽

Cited By ~ 1

Author(s):

Mika Tadokoro ◽

Noriko Kotobuki ◽

Akira Oshima ◽

Hajime Ohgushi

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

In Vitro Culture ◽

Porous Ceramic ◽

Porous Ceramics ◽

Human Mscs ◽

Ceramic Scaffold ◽

Before And After

This study focused on in vivo osteogenic capability of bone marrow mesenchymal stem cells (MSCs) seeded on ceramic scaffold. Human MSCs from a single donor were seeded on hydroxyapatite porous ceramic (HAP) and were induced to the osteogenic lineage during in vitro culture condition, then the MSCs/HAP composites were implanted subcutaneously into immunodeficient rats. The cellular activities of the composites were assayed in order to evaluate the distribution and differentiation capability of seeded MSCs before and after implantation. These results showed that the new bone, after implantation, was derived from the donor MSCs, which adhered to the surface of the ceramics pore areas during in vitro culture. Therefore, the engrafted donor cells proliferated and showed continuous osteogenic differentiation within the recipients. Consequently, our study demonstrates the usefulness of MSCs/HAP composites for clinical applications.

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Rheb1-Deficient Neutrophils Promote Hematopoietic Stem/Progenitor Cell Proliferation via Mesenchymal Stem Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.650599 ◽

2021 ◽

Vol 9 ◽

Author(s):

Juan Gao ◽

Shuaibing Hou ◽

Shengnan Yuan ◽

Yuxia Wang ◽

Yanan Gao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Myeloid Cells ◽

Human Mscs ◽

Hematopoietic Stem ◽

Progenitor Cell Proliferation ◽

High Level ◽

Lower Expression

Myeloid cells have been identified as hematopoietic stem cell (HSC)-regulating cells. However, the mechanisms by which myeloid cells regulate the function of HSCs are not fully defined. Our previous study indicated that the HSCs are over-expanded in Vav1-Cre;Rheb1fl/fl mice. Here, using in vivo and in vitro models, we found that Rheb1-deficient neutrophils remodeled the bone marrow environment and induced expansion of HSCs in vivo. Further studies showed that loss of Rheb1 impaired neutrophils’ ability to secrete IL-6, led mesenchymal stem cells (MSCs) to produce more SCF, and promote HSC proliferation. We further found that IL-6 suppressed SCF mRNA expression in human MSCs. Interesting, the high level of IL-6 was also related with poor survival of chronic myeloid leukemia (CML) patients, and higher expression of IL-6 in CML cells is associated with the lower expression of SCF in MSCs in patients. Our studies suggested that blocking IL-6 signaling pathway might stimulate MSCs to secrete more SCF, and to support hematopoietic stem/progenitor cells proliferation.

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Human Organoids for Predictive Toxicology Research and Drug Development

Frontiers in Genetics ◽

10.3389/fgene.2021.767621 ◽

2021 ◽

Vol 12 ◽

Author(s):

Toshikatsu Matsui ◽

Tadahiro Shinozawa

Keyword(s):

Stem Cells ◽

Drug Development ◽

Disease Modeling ◽

Cell Types ◽

Underlying Disease ◽

Future Research ◽

Specific Cell ◽

Predictive Toxicology

Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics.

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Vasculogenesis Potential of Mesenchymal and Endothelial Stem Cells Isolated from Various Human Tissues

10.1101/049668 ◽

2016 ◽

Cited By ~ 2

Author(s):

Rokhsareh Rohban ◽

Nathalie Etchart ◽

Thomas R. Pieber

Keyword(s):

Stem Cells ◽

Umbilical Cord ◽

Adult Stem Cell ◽

Cell Types ◽

Human Tissues ◽

Vessel Formation ◽

Nsg Mice ◽

Variable Capacity

AbstractNeo vessel formation can be initiated by co-transplantation of mesenchymal stem cells (MSC) with endothelial colony-forming cells (ECFC). The two adult stem cell types can be isolated and expanded from a variety of tissues to be used for regenerative applications pro-angiogenesis.Here we performed a systematic study to evaluate the neo-vasculogenesis potential of MSC and ECFC isolated from various human tissues. MSC were isolated, purified and expanded in vitro from umbilical cord (UC) and umbilical cord blood (UCB), white adipose tissue (WAT), bone marrow (BM), and amniotic membrane of placenta (AMN).ECFC were isolated from UC and UCB, WAT and peripheral blood (PB). ECFC and MSC and were co-transplanted admixed with extracellular matrix (Matrigel®) at a ratio of 5:1 to immune-deficient NSG mice, subcutaneously. The transplants were harvested after two weeks and the state of vessel formation and stability in the explants were investigated using immune-histochemical methods. The number of created micro-vessels was quantified using Hematoxylin & Eosin (H&E) staining followed by image J quantification.Results showed that ECFC and MSC possess variable capacity in contributing to neo-vasculogenesis. WAT and UCB-derived ECFC and WAT, UCB and BM-derived MSC are most potent cells in terms of neo-vessel formation in vivo. UC-derived ECFC and AMN-derived MSC have been shown to be least potent in contributing to neo-vasculogenesis. This variability might be due to variable phenotypes, or different genetic profiles of MSC and ECFC isolated from different tissues and/or donors.The findings might give an insight into better regenerative strategies for neo-vessel formation in vivo.

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