Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 459-469 ◽  
Author(s):  
Chih-Chien Tsai ◽  
Yann-Jang Chen ◽  
Tu-Lai Yew ◽  
Ling-Lan Chen ◽  
Jir-You Wang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hypoxia Inducible Factor ◽  
Human Mesenchymal Stem Cells ◽  
Telomerase Activity ◽  
Rapid Expansion ◽  
Low Density ◽  
Down Regulation ◽  
Differentiation Potential ◽  
Hypoxic Conditions ◽  
Population Doublings

Abstract Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor–1α (HIF-1α)–TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity.

scholarly journals Novel low shear 3D bioreactor for high purity mesenchymal stem cell production

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252575
Author(s):  
Andrew B. Burns ◽  
Corinna Doris ◽  
Kevin Vehar ◽  
Vinit Saxena ◽  
Cameron Bardliving ◽  
...  
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Stem Cell ◽  
High Purity ◽  
Culture Media ◽  
Human Mesenchymal Stem Cells ◽  
Three Dimensions ◽  
Bioreactor Culture ◽  
Differentiation Potential ◽  
Cell Culture Systems

Bone marrow derived human Mesenchymal Stem Cells (hMSCs) are an attractive candidate for regenerative medicine. However, their harvest can be invasive, painful, and expensive, making it difficult to supply the enormous amount of pure hMSCs needed for future allogeneic therapies. Because of this, a robust method of scaled bioreactor culture must be designed to supply the need for high purity, high density hMSC yields. Here we test a scaled down model of a novel bioreactor consisting of an unsubmerged 3D printed Polylactic Acid (PLA) lattice matrix wetted by culture media. The growth matrix is uniform, replicable, and biocompatible, enabling homogenous cell culture in three dimensions. The goal of this study was to prove that hMSCs would culture well in this novel bioreactor design. The system tested resulted in comparable stem cell yields to other cell culture systems using bone marrow derived hMSCs, while maintaining viability (96.54% ±2.82), high purity (>98% expression of combined positive markers), and differentiation potential.

Analyzing the behavior of normoxic and hypoxic cells through the use of microfluidic devices

2013 ◽  
Vol 6 (1) ◽  
Author(s):  
K. Chen ◽  
Rexius M. ◽  
D.T. Eddington
Keyword(s):  
Vascular Endothelial Cells ◽  
Experimental Testing ◽  
Hypoxia Inducible Factor ◽  
Human Mesenchymal Stem Cells ◽  
Microfluidic Devices ◽  
The Body ◽  
Cellular Growth ◽  
Hypoxic Conditions ◽  
Cellular Analysis ◽  
Growth Factor Production

Current cellular exposure to atmospheric normoxic (21%) oxygen concentrations have been proven to be physiologically inaccurate since the human body only ranges between 1%-13% in the body. We wish to observe how human mesenchymal stem cells (hMSCs) and human lung micro vascular endothelial cells (HLMVECs) interact with one another and their behavior when exposed to either hypoxic (defined as being less than normoxic) or atmospheric normoxic concentrations. The cells were grown and cultured on microfluidic devices - a relatively cheap and easily fabricated method of experimental testing that can lend itself to mass production and cellular analysis techniques. The analyses mainly focus on quantifying the amount of hypoxia-inducible factor-1 (HIF-1) present in the cells - this factor is responsible for activating countless transcription factors within the cell. Overall, these methods and tests have provided evidence to the fact that hypoxic conditions increase cellular growth, migration, proliferation, and growth factor production by almost two times.

scholarly journals Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity

2018 ◽  
Vol 74 (9) ◽  
pp. 1396-1407 ◽  
Author(s):  
Mujib Ullah ◽  
Zhongjie Sun
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Critical Role ◽  
Telomerase Activity ◽  
Cell Aging ◽  
Treated Animal ◽  
Differentiation Potential ◽  
Telomere Shortening ◽  
Altered Expression ◽  
Stem Cell Aging

Abstract Understanding the effect of molecular pathways involved in the age-dependent deterioration of stem cell function is critical for developing new therapies. The overexpression of Klotho (KL), an antiaging protein, causes treated animal models to enjoy extended life spans. Now, the question stands: Does KL deficiency accelerate stem cell aging and telomere shortening? If so, what are the specific mechanisms by which it does this, and is cycloastragenol (CAG) treatment enough to restore telomerase activity in aged stem cells? We found that KL deficiency diminished telomerase activity by altering the expression of TERF1 and TERT, causing impaired differentiation potential, pluripotency, cellular senescence, and apoptosis in stem cells. Telomerase activity decreased with KL-siRNA knockdown. This suggests that both KL and telomeres regulate the stem cell aging process through telomerase subunits TERF1, POT1, and TERT using the TGFβ, Insulin, and Wnt signaling. These pathways can rejuvenate stem cell populations in a CD90-dependent mechanism. Stem cell dysfunctions were largely provoked by KL deficiency and telomere shortening, owing to altered expression of TERF1, TGFβ1, CD90, POT1, TERT, and basic fibroblast growth factor (bFGF). The CAG treatment partially rescued telomerase deterioration, suggesting that KL plays a critical role in life-extension by regulating telomere length and telomerase activity.

scholarly journals Hypoxia-Inducible Factor 1α Induces Fibrosis and Insulin Resistance in White Adipose Tissue

2009 ◽  
Vol 29 (16) ◽  
pp. 4467-4483 ◽  
Author(s):  
Nils Halberg ◽  
Tayeba Khan ◽  
Maria E. Trujillo ◽  
Ingrid Wernstedt-Asterholm ◽  
Alan D. Attie ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Lysyl Oxidase ◽  
Active Form ◽  
Hypoxia Inducible Factor ◽  
Caloric Intake ◽  
Tissue Growth ◽  
Rapid Expansion ◽  
Hypoxia Inducible Factor 1Α ◽  
Early Stages ◽  
Hypoxic Conditions

ABSTRACT Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1α (HIF1α) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1α, we determined that HIF1α fails to induce the expected proangiogenic response. In contrast, we observed that HIF1α initiates adipose tissue fibrosis, with an associated increase in local inflammation. “Trichrome- and picrosirius red-positive streaks,” enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1α and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by β-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis.

scholarly journals Multi-differentiation potential is necessary for optimal tenogenesis of tendon stem cells

2019 ◽  
Author(s):  
Ibtesam Rajpar ◽  
Jennifer G. Barrett
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Cell Phenotype ◽  
Differentiation Potential ◽  
Therapeutic Benefits ◽  
Tendon Stem Cells ◽  
Stem Cell Lines ◽  
Population Doublings ◽  
Cell Phenotypes

ABSTRACTBackgroundTendon injury is a significant clinical problem, and regenerative treatments are limited by our understanding of endogenous tendon stem cells. Recent evidence suggests that tendon stem cells are diverse in phenotypic character, and may in fact exist on a spectrum of differentiation capacities. However, the functional significance of each differentiation phenotype is poorly understood. Toward this end, we performed a comprehensive assessment of differentiation capacity toward four connective tissue lineages (adipose, bone, cartilage and tendon) with clonal tendon stem cell lines to: 1) evaluate the differences, if any, in tenogenic potential, and 2) identify the relationships in differentiation phenotype and proliferation capacity.MethodsTendon stem cells were derived from whole equine flexor tendons for this study (N=3). Clonal tendon stem lines were generated by low-density cell plating, and subjected to standard assays of tri-lineage differentiation and population doublings. For tenogenesis, a previously engineered three-dimensional hydrogel construct was incorporated. Differentiation was quantified by the relative gene expression of lineage-specific markers, and confirmed with lineage-specific cell staining. Tenogenesis was further analyzed by hydrogel contraction and histomorphometry. Statistical significance was determined using analysis of variance and post-hoc Tukey’s tests.ResultsThree distinct tendon stem cell phenotypes were identified, namely differentiation toward: 1) adipose, bone, cartilage and tendon, 2) bone, cartilage and tendon, or 3) adipose, cartilage and tendon. Further, a positive correlation was found in the ability to differentiate toward all four lineages and the generation of a robust, composite tendon-like construct upon tenogenesis, manifested by the strongest expressions of scleraxis and mohawk, and parallel alignment of tenocyte-like cells with elongated cell morphologies. Significantly increased numbers of cumulative cell population doublings were seen in the absence of adipogenic potential in clonal tendon stem cell lines.ConclusionsOur study strengthens reports on the heterogeneous character of tendon stem cells and identifies key differences in their differentiation and proliferative potentials. Isolation of potent tendon stem cell populations from tendon stromal fractions may yield improved therapeutic benefits in clinical models of repair and promote a native, regenerative phenotype in engineered tendons. Future studies may be targeted to understanding the functional contributions of each tendon stem cell phenotype in vivo, and identifying additional cell phenotypes.

Multi-Potent Adult Progenitor Cells from Swine Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1704-1704
Author(s):  
Lepeng Zeng ◽  
Eric Rahrmann ◽  
Qingsong Hu ◽  
Xiaohong Wang ◽  
Jianyi Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Smooth Muscle ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Calcium Flux ◽  
Regulation Of Transcription ◽  
Differentiation Potential ◽  
Population Doublings

Abstract Here, we show that Multi-potent Adult Progenitor Cells (MAPCs) can be derived from adult and fetal swine bone marrow. Swine MAPC (swMAPC) contain initially multiple different progenitors and mature cells; however, cultures are homogenous by 50 population doublings and can grow past 150 population doublings. SwMAPCs are CD44, CD45, and MHC-I, II negative, express Oct3a/4 at levels close to those seen in embryonic stem cells, and have telomerase activity leading to no telomere shortening with expansion. We also have very solid evidence that swMAPCs differentiate into most mesoderm, neuroectoderm, and endoderm lineages as demonstrated by a significant up-regulation of transcription factors and other lineage specific proteins in a time dependent fashion similar to development, measured by Q-RT-PCR as well as immunohistology. In addition, swMAPCs induced to the endothelial lineage form vascular tubes, to the hepatic lineage produce albumin and urea, and to the smooth muscle lineage display significant calcium flux in response to smooth muscle agonists. In addition, we are investigating the swMAPC differentiation into cardiomyocytes. Preliminary data indicates swMAPCs express TBX5, Nkx2.5, and GATA6 by day 7 at significant levels. As we have seen for rodent MAPCs, when swMAPCs are allowed to grow at high density, Oct3a/4 levels drop to undetectable ranges, the cells take on a mesenchymal stem cell (MSC) phenotype, and the differentiation potential is lost. When replated under low density conditions, oct3a/4 expression or differentiation capacity can not be re-induced, and cells remain MSC like. Therefore, we suggest that Oct3a/4 is not an in vitro culture phenomena but is already present in swMAPCs and cannot be recovered once lost with standard culturing techniques.

Surface markers distinguishing mesenchymal stem cells from fibroblasts

2012 ◽  
Vol 19 (2) ◽  
pp. 75-79 ◽  
Author(s):  
Gabrielis KUNDROTAS
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Human Mesenchymal Stem Cells ◽  
Stem Cell Differentiation ◽  
Clinical Applications ◽  
Tumor Formation ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Differentiation Potential

Human mesenchymal stem cells (MSCs) are widely used for treatment of various diseases. Clinical applications require large quantities of MSCs, therefore these cells must be expanded in the culture system. It is believed that contamination of MSC cultures with fibroblasts may lead to the decrease of the stem cell differentiation potential. Moreover, such stem cell preparations are potentially unsafe to use for clinical applications since a few fibroblasts can become tumorigenic. Therefore, there is a need to separate MSCs from fibroblasts. However, studies show that MSCs and fibroblasts have much in common. These two types of cells share such properties as identical spindle-like morphology, plastic adherence and the same expression of most surface antigens. The aim of this review article is to analyze the literature on the similarities and differences between the MSCs and fibroblasts, particularly in the expression of cell surface markers in order to determine which could be used for quick separating of MSCs from fibroblasts. Interestingly, the results of recent studies suggest that the use of CD10, CD26, CD106, CD146 and ITGA11 could be helpful for the discrimination of MSCs from fibroblasts. Identification and elimination of fibroblasts from MSC cultures could improve the MSC yield and differentiation potential and also prevent possible tumor formation after MSC transplantation.

Metabolic Regulation and Related Molecular Mechanisms in Various Stem Cell Functions

2020 ◽  
Vol 15 (6) ◽  
pp. 531-546 ◽  
Author(s):  
Hwa-Yong Lee ◽  
In-Sun Hong
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Oxidative Phosphorylation ◽  
Metabolic Pathways ◽  
Critical Role ◽  
The Self ◽  
Specific Cell ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Cell Functions

Recent studies on the mechanisms that link metabolic changes with stem cell fate have deepened our understanding of how specific metabolic pathways can regulate various stem cell functions during the development of an organism. Although it was originally thought to be merely a consequence of the specific cell state, metabolism is currently known to play a critical role in regulating the self-renewal capacity, differentiation potential, and quiescence of stem cells. Many studies in recent years have revealed that metabolic pathways regulate various stem cell behaviors (e.g., selfrenewal, migration, and differentiation) by modulating energy production through glycolysis or oxidative phosphorylation and by regulating the generation of metabolites, which can modulate multiple signaling pathways. Therefore, a more comprehensive understanding of stem cell metabolism could allow us to establish optimal culture conditions and differentiation methods that would increase stem cell expansion and function for cell-based therapies. However, little is known about how metabolic pathways regulate various stem cell functions. In this context, we review the current advances in metabolic research that have revealed functional roles for mitochondrial oxidative phosphorylation, anaerobic glycolysis, and oxidative stress during the self-renewal, differentiation and aging of various adult stem cell types. These approaches could provide novel strategies for the development of metabolic or pharmacological therapies to promote the regenerative potential of stem cells and subsequently promote their therapeutic utility.

scholarly journals Let-7f miRNA regulates SDF-1α- and hypoxia-promoted migration of mesenchymal stem cells and attenuates mammary tumor growth upon exosomal release

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Virginia Egea ◽  
Kai Kessenbrock ◽  
Devon Lawson ◽  
Alexander Bartelt ◽  
Christian Weber ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Tumor Growth ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Human Mesenchymal Stem Cells ◽  
Target Cells ◽  
Autophagic Flux ◽  
Stromal Cell Derived Factor

AbstractBone marrow-derived human mesenchymal stem cells (hMSCs) are recruited to damaged or inflamed tissues where they contribute to tissue repair. This multi-step process involves chemokine-directed invasion of hMSCs and on-site release of factors that influence target cells or tumor tissues. However, the underlying molecular mechanisms are largely unclear. Previously, we described that microRNA let-7f controls hMSC differentiation. Here, we investigated the role of let-7f in chemotactic invasion and paracrine anti-tumor effects. Incubation with stromal cell-derived factor-1α (SDF-1α) or inflammatory cytokines upregulated let-7f expression in hMSCs. Transfection of hMSCs with let-7f mimics enhanced CXCR4-dependent invasion by augmentation of pericellular proteolysis and release of matrix metalloproteinase-9. Hypoxia-induced stabilization of the hypoxia-inducible factor 1 alpha in hMSCs promoted cell invasion via let-7f and activation of autophagy. Dependent on its endogenous level, let-7f facilitated hMSC motility and invasion through regulation of the autophagic flux in these cells. In addition, secreted let-7f encapsulated in exosomes was increased upon upregulation of endogenous let-7f by treatment of the cells with SDF-1α, hypoxia, or induction of autophagy. In recipient 4T1 tumor cells, hMSC-derived exosomal let-7f attenuated proliferation and invasion. Moreover, implantation of 3D spheroids composed of hMSCs and 4T1 cells into a breast cancer mouse model demonstrated that hMSCs overexpressing let-7f inhibited tumor growth in vivo. Our findings provide evidence that let-7f is pivotal in the regulation of hMSC invasion in response to inflammation and hypoxia, suggesting that exosomal let-7f exhibits paracrine anti-tumor effects.

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