Characterization of human adipose-derived stem cells

PURPOSE: There is a growing scientific interest in the plasticity and therapeutic potential of adipose-derived stem cells (ASCs), which are multipotent and abundant in adipose tissue and can differentiate in vitro into multiple lineages, including adipocytes, chondrocytes, osteoblasts, neural cells, endothelial cells and cardiomyocytes. The aim of this study was to isolate, cultivate and identify ASCs. METHODS: Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed. RESULTS: Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers). CONCLUSION: It was possible to isolate human adipose-derived stem cells by in vitro cultivation without adipogenic induction, maintaining their functional integrity and high proliferation levels. The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro.

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Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation

Cellular & Molecular Biology Letters ◽

10.2478/s11658-013-0083-2 ◽

2013 ◽

Vol 18 (2) ◽

Cited By ~ 16

Author(s):

Dana Foudah ◽

Juliana Redondo ◽

Cristina Caldara ◽

Fabrizio Carini ◽

Giovanni Tredici ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Therapeutic Potential ◽

Adipogenic Differentiation ◽

Neural Cells ◽

Human Mscs ◽

Differentiation Markers ◽

Neuronal Markers ◽

Wide Range

AbstractMesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.

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Probable impact of age and hypoxia on proliferation and microRNA expression profile of bone marrow-derived human mesenchymal stem cells

PeerJ ◽

10.7717/peerj.1536 ◽

2016 ◽

Vol 4 ◽

pp. e1536 ◽

Cited By ~ 18

Author(s):

Norlaily Mohd Ali ◽

Lily Boo ◽

Swee Keong Yeap ◽

Huynh Ky ◽

Dilan A. Satharasinghe ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Culture Condition ◽

Therapeutic Potential ◽

Age Groups ◽

Human Mesenchymal Stem Cells ◽

Culture Conditions ◽

Differentiation Potential

Decline in the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSC) is often seen with older donors as compared to young. Although hypoxia is known as an approach to improve the therapeutic potential of MSC in term of cell proliferation and differentiation capacity, its effects on MSC from aged donors have not been well studied. To evaluate the influence of hypoxia on different age groups, MSC from young (<30 years) and aged (>60 years) donors were expanded under hypoxic (5% O2) and normal (20% O2) culture conditions. MSC from old donors exhibited a reduction in proliferation rate and differentiation potential together with the accumulation of senescence features compared to that of young donors. However, MSC cultured under hypoxic condition showed enhanced self-renewing and proliferation capacity in both age groups as compared to normal condition. Bioinformatic analysis of the gene ontology (GO) and KEGG pathway under hypoxic culture condition identified hypoxia-inducible miRNAs that were found to target transcriptional activity leading to enhanced cell proliferation, migration as well as decrease in growth arrest and apoptosis through the activation of multiple signaling pathways. Overall, differentially expressed miRNA provided additional information to describe the biological changes of young and aged MSCs expansion under hypoxic culture condition at the molecular level. Based on our findings, the therapeutic potential hierarchy of MSC according to donor’s age group and culture conditions can be categorized in the following order: young (hypoxia) > young (normoxia) > old aged (hypoxia) > old aged (normoxia).

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Deregulated mito-nuclear communication alters chromatin plasticity and differentiation potential of mesenchymal stem cells upon ageing

10.1101/2020.04.02.022293 ◽

2020 ◽

Author(s):

Andromachi Pouikli ◽

Swati Parekh ◽

Monika Maleszewska ◽

Maarouf Baghdadi ◽

Ignacio Tripodi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Culture Conditions ◽

Osteogenic Potential ◽

Acetyl Coa ◽

Differentiation Potential ◽

General Decline ◽

Age Dependent ◽

Transcriptional Output

ABSTRACTAgeing is accompanied by a general decline in the function of many cellular pathways, with metabolic alterations, epigenetic modifications, and stem cell exhaustion representing three important hallmarks of the ageing process. However, whether these pathways are causally or functionally related at a molecular level remains poorly understood. Here, we use bone marrow-derived mesenchymal stem cells (MSCs) isolated from young and old mice to address how age-dependent changes in metabolism and epigenetics are linked and how they impact on the ageing transcriptome and differentiation potential. Given that MSCs maintain specific age-associated properties even under prolonged culture conditions, such as the age-dependent decrease in osteogenic differentiation, they are an excellent model to investigate in vitro the connection of ageing hallmarks on a mechanistic level. In this study, we demonstrate that upon ageing, osteogenic potential of MSCs declines as a consequence of deregulated mito-nuclear communication, mediated by decreased levels of the citrate carrier (CiC). Age-dependent down-regulation of CiC results in acetyl-CoA trapping within mitochondria, hypo-acetylation of histones and chromatin compaction. Together, these changes lead to an altered transcriptional output and are responsible for the reduced differentiation capacity into osteoblasts. Strikingly, short-term supplementation of aged cells with acetate, an exogenous source for cytosolic acetyl-CoA production, rescues not only the age-associated reduction of histone acetylation, but also the osteogenesis defect, representing a potential target for in vitro MSC rejuvenation.

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Towards Physiologic Culture Approaches to Improve Standard Cultivation of Mesenchymal Stem Cells

Cells ◽

10.3390/cells10040886 ◽

2021 ◽

Vol 10 (4) ◽

pp. 886

Author(s):

Ilias Nikolits ◽

Sabrina Nebel ◽

Dominik Egger ◽

Sebastian Kreß ◽

Cornelia Kasper

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

In Vitro Culture ◽

Culture Media ◽

Culture Conditions ◽

Primary Phase ◽

Current Standard ◽

Differentiation Potential ◽

Therapeutic Uses

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.

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Probable impact of age and hypoxia on proliferation and microRNA expression profile of bone marrow-derived human mesenchymal stem cells

10.7287/peerj.preprints.1526v1 ◽

2015 ◽

Author(s):

Norlaily Mohd Ali ◽

Lily Boo ◽

Swee Keong Yeap ◽

Huynh Ky ◽

Dilan A Satharasinghe ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Culture Condition ◽

Therapeutic Potential ◽

Age Groups ◽

Human Mesenchymal Stem Cells ◽

Culture Conditions ◽

Differentiation Potential

Decline in the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSC) is often seen with older donors as compared to young. Although hypoxia is known as an approach to improve the therapeutic potential of MSC in term of cell proliferation and differentiation capacity, its effects on MSC from aged donors have not been well studied. To evaluate the influence of hypoxia on different age groups, MSC from young (<30 years) and aged (>60 years) donors were expanded under hypoxic (5% O2) and normal (20% O2) culture conditions. MSC from old donors exhibited a reduction in proliferation rate and differentiation potential together with the accumulation of senescence features compared to that of young donors. However, MSC cultured under hypoxic condition showed enhanced self-renewing and proliferation capacity in both age groups as compared to normal condition. Bioinformatic analysis of the gene ontology (GO) and KEGG pathway under hypoxic culture condition identified hypoxia-inducible miRNAs that were found to target transcriptional activity leading to enhanced cell proliferation, migration as well as decrease in growth arrest and apoptosis through the activation of multiple signaling pathways. Overall, differentially expressed miRNA provided additional information to describe the biological changes of young and aged MSCs expansion under hypoxic culture condition at the molecular level. Based on our findings, the therapeutic potential hierarchy of MSC according to donor’s age group and culture conditions can be categorized in the following order: young (hypoxia)> young (normoxia) > old aged (hypoxia) > old aged (normoxia).

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Probable impact of age and hypoxia on proliferation and microRNA expression profile of bone marrow-derived human mesenchymal stem cells

10.7287/peerj.preprints.1526 ◽

2015 ◽

Author(s):

Norlaily Mohd Ali ◽

Lily Boo ◽

Swee Keong Yeap ◽

Huynh Ky ◽

Dilan A Satharasinghe ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Culture Condition ◽

Therapeutic Potential ◽

Age Groups ◽

Human Mesenchymal Stem Cells ◽

Culture Conditions ◽

Differentiation Potential

Decline in the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSC) is often seen with older donors as compared to young. Although hypoxia is known as an approach to improve the therapeutic potential of MSC in term of cell proliferation and differentiation capacity, its effects on MSC from aged donors have not been well studied. To evaluate the influence of hypoxia on different age groups, MSC from young (<30 years) and aged (>60 years) donors were expanded under hypoxic (5% O2) and normal (20% O2) culture conditions. MSC from old donors exhibited a reduction in proliferation rate and differentiation potential together with the accumulation of senescence features compared to that of young donors. However, MSC cultured under hypoxic condition showed enhanced self-renewing and proliferation capacity in both age groups as compared to normal condition. Bioinformatic analysis of the gene ontology (GO) and KEGG pathway under hypoxic culture condition identified hypoxia-inducible miRNAs that were found to target transcriptional activity leading to enhanced cell proliferation, migration as well as decrease in growth arrest and apoptosis through the activation of multiple signaling pathways. Overall, differentially expressed miRNA provided additional information to describe the biological changes of young and aged MSCs expansion under hypoxic culture condition at the molecular level. Based on our findings, the therapeutic potential hierarchy of MSC according to donor’s age group and culture conditions can be categorized in the following order: young (hypoxia)> young (normoxia) > old aged (hypoxia) > old aged (normoxia).

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Comparative Study on In Vitro Culture of Mouse Bone Marrow Mesenchymal Stem Cells

Stem Cells International ◽

10.1155/2018/6704583 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 12

Author(s):

Yuxin Hu ◽

Bin Lou ◽

Xiafang Wu ◽

Ruirui Wu ◽

Huihui Wang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Culture Method ◽

Culture Conditions ◽

Mouse Bone Marrow ◽

Differentiation Potential ◽

Initial Medium

In vitro culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) has been hampered because of the low yield of MSCs during isolation and the contamination of hematopoietic cells during expansion. The lack of specific mouse BM-MSC markers increases the difficulty. Several techniques have been reported to improve the purity and in vitro growth of mouse BM-MSCs. However, systematic report on comparison of characteristics in primary BM-MSCs between different culture conditions is rare. Here, we studied the effects of oxygen concentrations and initial medium replacement intervals, along with cell passages, on mouse BM-MSCs isolated with differential adhesion method. BM-MSCs exhibited elevated proliferative and clonogenic abilities in 5% oxygen compared with 10% and 21% oxygen, as well as a better expression of the MSC marker Sca-1. Adipogenic and osteogenetic differentiation of BM-MSCs can be observed in both 21% and 5% oxygen. Adipogenic differentiation appeared stronger under normoxia conditions. BM-MSCs showed increased proliferative capacity and adipogenic/osteogenetic differentiation potential when initial medium replacement interval was 4 days compared with 1 day. As passage number increased, cells were more MSC-like in morphology and in expression of surface markers (positive for CD29, CD44, and Sca-1 and negative for CD11b, CD19, and CD45). These data provide new insight into optimizing the culture method and understanding the biological characteristics of mouse BM-MSCs during in vitro expansion.

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Cyclic Strain and Electrical Co-stimulation Improve Neural Differentiation of Marrow-Derived Mesenchymal Stem Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.624755 ◽

2021 ◽

Vol 9 ◽

Author(s):

Hong Cheng ◽

Yan Huang ◽

Wei Chen ◽

Jifei Che ◽

Taidong Liu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Electrical Stimulation ◽

Growth Factor ◽

Neural Differentiation ◽

Cyclic Strain ◽

Stem Cell Differentiation ◽

Neural Cells ◽

Differentiation Potential

The current study investigated the combinatorial effect of cyclic strain and electrical stimulation on neural differentiation potential of rat bone marrow-derived mesenchymal stem cells (BMSCs) under epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) inductions in vitro. We developed a prototype device which can provide cyclic strain and electrical signal synchronously. Using this system, we demonstrated that cyclic strain and electrical co-stimulation promote the differentiation of BMCSs into neural cells with more branches and longer neurites than strain or electrical stimulation alone. Strain and electrical co-stimulation can also induce a higher expression of neural markers in terms of transcription and protein level. Neurotrophic factors and the intracellular cyclic AMP (cAMP) are also upregulated with co-stimulation. Importantly, the co-stimulation further enhances the calcium influx of neural differentiated BMSCs when responding to acetylcholine and potassium chloride (KCl). Finally, the phosphorylation of extracellular-signal-regulated kinase (ERK) 1 and 2 and protein kinase B (AKT) was elevated under co-stimulation treatment. The present work suggests a synergistic effect of the combination of cyclic strain and electrical stimulation on BMSC neuronal differentiation and provides an alternative approach to physically manipulate stem cell differentiation into mature and functional neural cells in vitro.

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Therapeutic Potential of Amniotic Fluid Derived Mesenchymal Stem Cells Based on their Differentiation Capacity and Immunomodulatory Properties

Current Stem Cell Research & Therapy ◽

10.2174/1574888x14666190222201749 ◽

2019 ◽

Vol 14 (4) ◽

pp. 327-336 ◽

Cited By ~ 9

Author(s):

Carl R. Harrell ◽

Marina Gazdic ◽

Crissy Fellabaum ◽

Nemanja Jovicic ◽

Valentin Djonov ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Animal Models ◽

Amniotic Fluid ◽

Therapeutic Potential ◽

Inflammatory Diseases ◽

Therapeutic Effects ◽

Differentiation Potential ◽

Differentiation Capacity ◽

Cell Based Therapy

Background: Amniotic Fluid Derived Mesenchymal Stem Cells (AF-MSCs) are adult, fibroblast- like, self-renewable, multipotent stem cells. During the last decade, the therapeutic potential of AF-MSCs, based on their huge differentiation capacity and immunomodulatory characteristics, has been extensively explored in animal models of degenerative and inflammatory diseases. Objective: In order to describe molecular mechanisms responsible for the therapeutic effects of AFMSCs, we summarized current knowledge about phenotype, differentiation potential and immunosuppressive properties of AF-MSCs. Methods: An extensive literature review was carried out in March 2018 across several databases (MEDLINE, EMBASE, Google Scholar), from 1990 to present. Keywords used in the selection were: “amniotic fluid derived mesenchymal stem cells”, “cell-therapy”, “degenerative diseases”, “inflammatory diseases”, “regeneration”, “immunosuppression”. Studies that emphasized molecular and cellular mechanisms responsible for AF-MSC-based therapy were analyzed in this review. Results: AF-MSCs have huge differentiation and immunosuppressive potential. AF-MSCs are capable of generating cells of mesodermal origin (chondrocytes, osteocytes and adipocytes), neural cells, hepatocytes, alveolar epithelial cells, insulin-producing cells, cardiomyocytes and germ cells. AF-MSCs, in juxtacrine or paracrine manner, regulate proliferation, activation and effector function of immune cells. Due to their huge differentiation capacity and immunosuppressive characteristic, transplantation of AFMSCs showed beneficent effects in animal models of degenerative and inflammatory diseases of nervous, respiratory, urogenital, cardiovascular and gastrointestinal system. Conclusion: Considering the fact that amniotic fluid is obtained through routine prenatal diagnosis, with minimal invasive procedure and without ethical concerns, AF-MSCs represents a valuable source for cell-based therapy of organ-specific or systemic degenerative and inflammatory diseases.

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