scholarly journals Hypoxic Culture Conditions as a Solution for Mesenchymal Stem Cell Based Regenerative Therapy

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Nazmul Haque ◽  
Mohammad Tariqur Rahman ◽  
Noor Hayaty Abu Kasim ◽  
Aied Mohammed Alabsi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Growth Kinetics ◽  
Chemokine Receptors ◽  
Hypoxia Inducible Factor ◽  
Culture Conditions ◽  
Poor Growth ◽  
Regenerative Therapies

Cell-based regenerative therapies, based onin vitropropagation of stem cells, offer tremendous hope to many individuals suffering from degenerative diseases that were previously deemed untreatable. Due to the self-renewal capacity, multilineage potential, and immunosuppressive property, mesenchymal stem cells (MSCs) are considered as an attractive source of stem cells for regenerative therapies. However, poor growth kinetics, early senescence, and genetic instability duringin vitroexpansion and poor engraftment after transplantation are considered to be among the major disadvantages of MSC-based regenerative therapies. A number of complex inter- and intracellular interactive signaling systems control growth, multiplication, and differentiation of MSCs in their niche. Common laboratory conditions for stem cell culture involve ambient O2concentration (20%) in contrast to their niche where they usually reside in 2–9% O2. Notably, O2plays an important role in maintaining stem cell fate in terms of proliferation and differentiation, by regulating hypoxia-inducible factor-1 (HIF-1) mediated expression of different genes. This paper aims to describe and compare the role of normoxia (20% O2) and hypoxia (2–9% O2) on the biology of MSCs. Finally it is concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and expression of chemokine receptors duringin vitroexpansion and eventually can increase efficiency of MSC-based regenerative therapies.

scholarly journals Regulation and Directing Stem Cell Fate by Tissue Engineering Functional Microenvironments: Scaffold Physical and Chemical Cues

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Fei Xing ◽  
Lang Li ◽  
Changchun Zhou ◽  
Cheng Long ◽  
Lina Wu ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Cell Fate ◽  
Chemical Cues ◽  
Physical Factors ◽  
Stem Cell Fate ◽  
Physical And Chemical

It is well known that stem cells reside within tissue engineering functional microenvironments that physically localize them and direct their stem cell fate. Recent efforts in the development of more complex and engineered scaffold technologies, together with new understanding of stem cell behavior in vitro, have provided a new impetus to study regulation and directing stem cell fate. A variety of tissue engineering technologies have been developed to regulate the fate of stem cells. Traditional methods to change the fate of stem cells are adding growth factors or some signaling pathways. In recent years, many studies have revealed that the geometrical microenvironment played an essential role in regulating the fate of stem cells, and the physical factors of scaffolds including mechanical properties, pore sizes, porosity, surface stiffness, three-dimensional structures, and mechanical stimulation may affect the fate of stem cells. Chemical factors such as cell-adhesive ligands and exogenous growth factors would also regulate the fate of stem cells. Understanding how these physical and chemical cues affect the fate of stem cells is essential for building more complex and controlled scaffolds for directing stem cell fate.

scholarly journals Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1873 ◽  
Author(s):  
Andrea Remuzzi ◽  
Barbara Bonandrini ◽  
Matteo Tironi ◽  
Lorena Longaretti ◽  
Marina Figliuzzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Cultured Cells ◽  
Three Dimensions ◽  
Nuclear Pore ◽  
Cells Cultured

Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the “Nichoid”, an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or “2D Nichoid” and multi-layered or “3D Nichoid”) were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells’ specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells’ features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo.

Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3546-3546
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Andre Larochelle ◽  
Nancy F. Hensel ◽  
Keyvan Keyvanfar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Culture Conditions ◽  
Myelogenous Leukemia ◽  
Cell Cycle Analysis ◽  
Remission Induction ◽  
Leukemia Stem Cell

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Constructing stem cell microenvironments using bioengineering approaches

2013 ◽  
Vol 45 (23) ◽  
pp. 1123-1135 ◽  
Author(s):  
David A. Brafman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Disease Modeling ◽  
Mechanical Stimuli ◽  
Culture Methods ◽  
Stem Cell Fate ◽  
And Function

Within the adult organism, stem cells reside in defined anatomical microenvironments called niches. These architecturally diverse microenvironments serve to balance stem cell self-renewal and differentiation. Proper regulation of this balance is instrumental to tissue repair and homeostasis, and any imbalance can potentially lead to diseases such as cancer. Within each of these microenvironments, a myriad of chemical and physical stimuli interact in a complex (synergistic or antagonistic) manner to tightly regulate stem cell fate. The in vitro replication of these in vivo microenvironments will be necessary for the application of stem cells for disease modeling, drug discovery, and regenerative medicine purposes. However, traditional reductionist approaches have only led to the generation of cell culture methods that poorly recapitulate the in vivo microenvironment. To that end, novel engineering and systems biology approaches have allowed for the investigation of the biological and mechanical stimuli that govern stem cell fate. In this review, the application of these technologies for the dissection of stem cell microenvironments will be analyzed. Moreover, the use of these engineering approaches to construct in vitro stem cell microenvironments that precisely control stem cell fate and function will be reviewed. Finally, the emerging trend of using high-throughput, combinatorial methods for the stepwise engineering of stem cell microenvironments will be explored.

Epigenetic regulation in stem cell development, cell fate conversion, and reprogramming

2015 ◽  
Vol 6 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Kazuyuki Ohbo ◽  
Shin-ichi Tomizawa
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Cell Fate ◽  
Cell Lines ◽  
Cell Fate Decisions ◽  
Micro Rnas ◽  
Stem Cell Lines ◽  
Cell Fate Conversion

AbstractStem cells are identified classically by an in vivo transplantation assay plus additional characterization, such as marker analysis, linage-tracing and in vitro/ex vivo differentiation assays. Stem cell lines have been derived, in vitro, from adult tissues, the inner cell mass (ICM), epiblast, and male germ stem cells, providing intriguing insight into stem cell biology, plasticity, heterogeneity, metastable state, and the pivotal point at which stem cells irreversibly differentiate to non-stem cells. During the past decade, strategies for manipulating cell fate have revolutionized our understanding about the basic concept of cell differentiation: stem cell lines can be established by introducing transcription factors, as with the case for iPSCs, revealing some of the molecular interplay of key factors during the course of phenotypic changes. In addition to de-differentiation approaches for establishing stem cells, another method has been developed whereby induced expression of certain transcription factors and/or micro RNAs artificially converts differentiated cells from one committed lineage to another; notably, these cells need not transit through a stem/progenitor state. The molecular cues guiding such cell fate conversion and reprogramming remain largely unknown. As differentiation and de-differentiation are directly linked to epigenetic changes, we overview cell fate decisions, and associated gene and epigenetic regulations.

scholarly journals Morphogen And Community Effects Determine Cell Fates In Response To BMP4 Signaling In Human Embryonic Stem Cells

2017 ◽  
Author(s):  
Anastasiia Nemashkalo ◽  
Albert Ruzo ◽  
Idse Heemskerk ◽  
Aryeh Warmflash
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Community Effects ◽  
Cell Fates ◽  
Standard Culture

AbstractParacrine signals maintain developmental states and create cell-fate patterns in vivo, and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro. Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells (“μColonies”) to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in μColonies and standard culture conditions and find that in μColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions, BMP4 acts as morphogen, but this effect requires secondary signals and particular cell densities. We further find that a “community effect” enforces a common fate within μColonies both in the state of pluripotency and when cells are differentiated, and that this effect allows more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.Summary StatementWe quantitatively examined signaling and differentiation in hESC colonies of varying size treated with BMP4. We show that secondary signals result in morphogen and community effects that determine cell fates.

Abstract 213: Evolving Challenges in Promoting Stem Cell Based Cardiovascular Repair and Regeneration

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Mani T Valarmathi ◽  
Jiang Li
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cardiac Muscle ◽  
Adult Stem Cells ◽  
Cardiac Tissue ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Myocardial Damage ◽  
Culture Conditions

Introduction: Use of adult stem cells in the stimulation of mammalian cardiac muscle regeneration is in its infancy, and to date, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone-marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific cells, and also exhibit functional synchronization. As a result, this necessitates the development of an appropriate in vitro three-dimensional (3-D) model of cardiomyogenesis and prompts the development of a 3-D cardiac muscle construct for tissue engineering purposes, especially using the adult stem cells. Hypothesis: Functioning vascularized cardiac tissue can be generated by the interaction of human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human multipotent mesenchymal stem cells (hMSCs) on a 3-D prevascularized collagen cell carrier (CCC) scaffold. Methods and Results: In order to achieve the above aim, we have developed an in vitro 3-D functioning vascularized cardiac muscle construct using hiPSC-ECMs and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured on 3-D CCCs for 7 days under vasculogenic culture conditions, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of micro vessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed dramatic neo-angiogenesis and neo-cardiomyogenesis. Conclusions: Thus, our unique 3-D co-culture system provided us the apt in vitro functioning prevascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.

scholarly journals Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Sébastien Sart ◽  
Liqing Song ◽  
Yan Li
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Disease Modeling ◽  
Redox Status ◽  
Culture Conditions ◽  
Ros Generation ◽  
Cellular Functions ◽  
Proliferation And Differentiation ◽  
Cell Organization

Reactive oxygen species (ROS) have long been considered as pathological agents inducing apoptosis under adverse culture conditions. However, recent findings have challenged this dogma and physiological levels of ROS are now considered as secondary messengers, mediating numerous cellular functions in stem cells. Stem cells represent important tools for tissue engineering, drug screening, and disease modeling. However, the safe use of stem cells for clinical applications still requires culture improvements to obtain functional cells. With the examples of mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs), this review investigates the roles of ROS in the maintenance of self-renewal, proliferation, and differentiation of stem cells. In addition, this work highlights that the tight control of stem cell microenvironment, including cell organization, and metabolic and mechanical environments, may be an effective approach to regulate endogenous ROS generation. Taken together, this paper indicates the need for better quantification of ROS towards the accurate control of stem cell fate.

scholarly journals Controlling Self-Renewal and Differentiation of Stem Cells via Mechanical Cues

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Michele M. Nava ◽  
Manuela T. Raimondi ◽  
Riccardo Pietrabissa
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Cell Response ◽  
Recent Literature ◽  
Focal Adhesions ◽  
Substrate Stiffness ◽  
Self Renewal ◽  
Mechanical Factors

The control of stem cell responsein vitro, including self-renewal and lineage commitment, has been proved to be directed by mechanical cues, even in the absence of biochemical stimuli. Through integrin-mediated focal adhesions, cells are able to anchor onto the underlying substrate, sense the surrounding microenvironment, and react to its properties. Substrate-cell and cell-cell interactions activate specific mechanotransduction pathways that regulate stem cell fate. Mechanical factors, including substrate stiffness, surface nanotopography, microgeometry, and extracellular forces can all have significant influence on regulating stem cell activities. In this paper, we review all the most recent literature on the effect of purely mechanical cues on stem cell response, and we introduce the concept of “force isotropy” relevant to cytoskeletal forces and relevant to extracellular loads acting on cells, to provide an interpretation of how the effects of insoluble biophysical signals can be used to direct stem cells fatein vitro.

scholarly journals An in vitro stem cell model of human epiblast and yolk sac interaction

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kirsty ML Mackinlay ◽  
Bailey AT Weatherbee ◽  
Viviane Souza Rosa ◽  
Charlotte E Handford ◽  
George Hudson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Human Embryo ◽  
Cell Model ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryonic Cells ◽  
Post Implantation

Human embryogenesis entails complex signalling interactions between embryonic and extra-embryonic cells. However, how extra-embryonic cells direct morphogenesis within the human embryo remains largely unknown due to a lack of relevant stem cell models. Here, we have established conditions to differentiate human pluripotent stem cells (hPSCs) into yolk sac-like cells (YSLCs) that resemble the post-implantation human hypoblast molecularly and functionally. YSLCs induce the expression of pluripotency and anterior ectoderm markers in human embryonic stem cells (hESCs) at the expense of mesoderm and endoderm markers. This activity is mediated by the release of BMP and WNT signalling pathway inhibitors, and, therefore, resembles the functioning of the anterior visceral endoderm signalling centre of the mouse embryo, which establishes the anterior-posterior axis. Our results implicate the yolk sac in epiblast cell fate specification in the human embryo and propose YSLCs as a tool for studying post-implantation human embryo development in vitro.

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