scholarly journals Use of conditioned media (CM) and xeno-free serum substitute on human adipose-derived stem cells (ADSCs) differentiation into urothelial-like cells

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10890
Author(s):  
Ban Al- kurdi ◽  
Nidaa A. Ababneh ◽  
Nizar Abuharfeil ◽  
Saddam Al Demour ◽  
Abdalla S. Awidi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Urinary Tract ◽  
Culture Media ◽  
Urothelial Cell ◽  
Conditioned Media ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Expression Levels

Background Congenital abnormalities, cancers as well as injuries can cause irreversible damage to the urinary tract, which eventually requires tissue reconstruction. Smooth muscle cells, endothelial cells, and urothelial cells are the major cell types required for the reconstruction of lower urinary tract. Adult stem cells represent an accessible source of unlimited repertoire of untransformed cells. Aim Fetal bovine serum (FBS) is the most vital supplement in the culture media used for cellular proliferation and differentiation. However, due to the increasing interest in manufacturing xeno-free stem cell-based cellular products, optimizing the composition of the culture media and the serum-type used is of paramount importance. In this study, the effects of FBS and pooled human platelet (pHPL) lysate were assessed on the capacity of human adipose-derived stem cells (ADSCs) to differentiate into urothelial-like cells. Also, we aimed to compare the ability of both conditioned media (CM) and unconditioned urothelial cell media (UCM) to induce urothelial differentiation of ADCS in vitro. Methods ADSCs were isolated from human lipoaspirates and characterized by flow cytometry for their ability to express the most common mesenchymal stem cell (MSCs) markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of ADSCs to differentiate towards the urothelial-like lineage, cells were cultured with either CM or UCM, supplemented with either 5% pHPL, 2.5% pHPL or 10% FBS. After 14 days of induction, cells were utilized for gene expression and immunofluorescence analysis. Results ADSCs cultured in CM and supplemented with FBS exhibited the highest upregulation levels of the urothelial cell markers; cytokeratin-18 (CK-18), cytokeratin-19 (CK-19), and Uroplakin-2 (UPK-2), with a 6.7, 4.2- and a 2-folds increase in gene expression, respectively. Meanwhile, the use of CM supplemented with either 5% pHPL or 2.5% pHPL, and UCM supplemented with either 5% pHPL or 2.5% pHPL showed low expression levels of CK-18 and CK-19 and no upregulation of UPK-2 level was observed. In contrast, the use of UCM with FBS has increased the levels of CK-18 and CK-19, however to a lesser extent compared to CM. At the cellular level, CK-18 and UPK-2 were only detected in CM/FBS supplemented group. Growth factor analysis revealed an increase in the expression levels of EGF, VEGF and PDGF in all of the differentiated groups. Conclusion Efficient ADSCs urothelial differentiation is dependent on the use of conditioned media. The presence of high concentrations of proliferation-inducing growth factors present in the pHPL reduces the efficiency of ADSCs differentiation towards the urothelial lineage. Additionally, the increase in EGF, VEGF and PDGF during the differentiation implicates them in the mechanism of urothelial cell differentiation.

scholarly journals Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Gayathri Subramanian ◽  
Alexander Stasuk ◽  
Mostafa Elsaadany ◽  
Eda Yildirim-Ayan
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Uniaxial Tensile ◽  
Adipose Derived Stem Cells ◽  
Collagen Scaffolds ◽  
Differentiation Potential ◽  
Tenogenic Differentiation ◽  
Matrix Organization

Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment.

scholarly journals The Effect of Blood-Derived Products on the Chondrogenic and Osteogenic Differentiation Potential of Adipose-Derived Mesenchymal Stem Cells Originated from Three Different Locations

2019 ◽  
Vol 2019 ◽  
pp. 1-20 ◽  
Author(s):  
Markus Neubauer ◽  
Olga Kuten ◽  
Christoph Stotter ◽  
Karina Kramer ◽  
Andrea De Luna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Blood Product ◽  
Blood Products ◽  
Fat Tissue ◽  
Differentiation Potential ◽  
Lower Blood

Background. Adipose-derived mesenchymal stem cells (AD-MSCs) from fat tissue considered “surgical waste” during joint surgery may provide a potent source for regenerative medicine. Intra-articular, homologous fat tissue (Hoffa’s fat pad, pouch fat) might possess a superior chondrogenic and osteogenic differentiation potential in comparison to extra-articular, nonhomologous fat. Blood products might further enhance this potential. Methods. AD-MSCs were isolated from fat tissue of 3 donors from 3 locations each, during total knee replacement. Isolated cells were analyzed via flow cytometry. Cells were supplemented with blood products: two types of platelet-rich plasma (EPRP—PRP prepared in the presence of EDTA; CPRP—PRP prepared in the presence of citrate), hyperacute serum (hypACT), and standard fetal calf serum (FCS) as a positive control. The viability of the cells was determined by XTT assay, and the progress of differentiation was tested via histological staining and monitoring of specific gene expression. Results. Blood products enhance ex vivo cell metabolism. Chondrogenesis is enhanced by EDTA-PRP and osteogenesis by citrate PRP, whereas hyperacute serum enhances both differentiations comparably. This finding was consistent in histological analysis as well as in gene expression. Lower blood product concentrations and shorter differentiation periods lead to superior histological results for chondrogenesis. Both PRP types had a different biological effect depending upon concentration, whereas hyperacute serum seemed to have a more consistent effect, independent of the used concentration. Conclusion. (i) Blood product preparation method, (ii) type of anticoagulant, (iii) differentiation time, and (iv) blood product concentration have a significant influence on stem cell viability and the differentiation potential, favouring no use of anticoagulation, shorter differentiation time, and lower blood product concentrations. Cell-free blood products like hyperacute serum may be considered as an alternative supplementation in regenerative medicine, especially for stem cell therapies.

scholarly journals Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Aistė Zentelytė ◽  
Deimantė Žukauskaitė ◽  
Ieva Jacerytė ◽  
Veronika V. Borutinskaitė ◽  
Rūta Navakauskienė
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Amniotic Fluid ◽  
Small Molecules ◽  
Small Molecule ◽  
Human Amniotic Fluid ◽  
Short Term ◽  
Differentiation Potential ◽  
Amniotic Fluid Stem Cells

Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.

scholarly journals Integrin-α3 Is a New Marker of Long-Term Hematopoietic Stem Cells in Ex Vivo Expanded Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1267-1267
Author(s):  
Elisa Tomellini ◽  
Iman Fares ◽  
Bernhard Lehnertz ◽  
Jalila Chagraoui ◽  
Nadine Mayotte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stem Cell Markers ◽  
Short Term ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell (HSC) transplantation constitutes one of the most effective therapeutic strategies to treat numerous hematological diseases. Cord blood (CB) is one of the most attractive donor sources of stem cells for this procedure due to its rapid availability, HLA mismatches tolerance and low associated risk of chronic graft-versus-host disease. However, these advantages are offset by the limited cell dose in CB units, which can contribute to delayed hematopoietic engraftment following transplantation. Mastering ex vivo HSC expansion is therefore of great interest for clinical purposes and for genetic manipulation. HSCs can be functionally defined as either long-term (LT-HSC), providing life-long hematopoiesis and characterized by delayed engraftment pattern, or short-term repopulating stem cells (ST-HSC), providing early and transient hematopoietic recovery. Major hurdles hindering the study of these cell populations is the current inability to evaluate their content in cultured samples and the lack of understanding of the molecular mechanisms regulating stem cell self-renewal ex vivo. Those issues highly benefited from the discovery by our laboratory of the small molecule UM171, which promote HSC expansion ex vivo, as well as from the identification of EPCR as one of the most reliable surface markers for cultured HSCs. We now describe the identification of Integrin-α3 (ITGA3) as a novel marker for cultured HSCs. ITGA3 expression was found to be sufficient to split the primitive EPCR+CD90+CD133+CD34+CD45RA- HSC population in two functionally distinct fractions presenting only short-term (ITGA3-) and both short-term and long-term (ITGA3+) repopulating potential. ITGA3+ cells, as opposed to the ITGA3- fraction, exhibited robust multilineage differentiation potential and serial reconstitution ability in immunocompromised mice. This combination of markers identifies repopulating HSCs in culture by FACS beyond what is currently possible with other approaches, with a frequency of LT-HSC found in the ITGA3+ population estimated at 1:38 in day 7 UM171 expanded CB-cells. Moreover, lentiviral-mediated ITGA3 knockdown was shown to compromise the LT repopulating activity of cultured HSC in vivo. Gene expression profiling revealed striking molecular similarity between ITGA3+ and ITGA3- cells, showing overrepresentation of genes involved in fundamental hematopoietic programs known to govern HSC specification and function in both of these populations. However, ITGA3+ and ITGA3- subsets clearly clustered separately by principle component analysis, indicating broad differences in gene expression. Concordantly with their primitive phenotype, stem cell markers and cell quiescence are gene sets enriched in ITGA3+ cells, while progenitor markers, DNA replication, M/G1 and G2/M checkpoints, mRNA processing, reduction of hypoxia and Myc targets were significantly downregulated in these cells. Altogether, our results indicate that ITGA3 is a reliable marker for cultured HSCs, improving the accuracy of prospective HSC identification in culture. Deciphering the function of genes upregulated in primitive ITGA3+ HSCs will represent an invaluable resource for dissecting the genetic programs that govern hematopoietic stem cells biology. Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

scholarly journals CD10 marks non-canonical PPARγ-independent adipocyte maturation and browning potential of adipose-derived stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Smarajit Chakraborty ◽  
Wee Kiat Ong ◽  
Winifred W. Y. Yau ◽  
Zhihong Zhou ◽  
K. N. Bhanu Prakash ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Human Subject ◽  
Metabolic Diseases ◽  
Quantitative Polymerase Chain Reaction ◽  
Target Cells ◽  
Peroxisome Proliferator ◽  
Adipose Derived Stem Cells ◽  
Direct Role ◽  
Differentiation Potential

Abstract Background Effective stem cell therapy is dependent on the stem cell quality that is determined by their differentiation potential, impairment of which leads to poor engraftment and survival into the target cells. However, limitations in our understanding and the lack of reliable markers that can predict their maturation efficacies have hindered the development of stem cells as an effective therapeutic strategy. Our previous study identified CD10, a pro-adipogenic, depot-specific prospective cell surface marker of human adipose-derived stem cells (ASCs). Here, we aim to determine if CD10 can be used as a prospective marker to predict mature adipocyte quality and play a direct role in adipocyte maturation. Methods We first generated 14 primary human subject-derived ASCs and stable immortalized CD10 knockdown and overexpression lines for 4 subjects by the lentiviral transduction system. To evaluate the role of CD10 in adipogenesis, the adipogenic potential of the human subject samples were scored against their respective CD10 transcript levels. Assessment of UCP1 expression levels was performed to correlate CD10 levels to the browning potential of mature ASCs. Quantitative polymerase chain reaction (qPCR) and Western blot analysis were performed to determine CD10-dependent regulation of various targets. Seahorse analysis of oxidative metabolism and lipolysis assay were studied. Lastly, as a proof-of-concept study, we used CD10 as a prospective marker for screening nuclear receptor ligands library. Results We identified intrinsic CD10 levels as a positive determinant of adipocyte maturation as well as browning potential of ASCs. Interestingly, CD10 regulates ASC’s adipogenic maturation non-canonically by modulating endogenous lipolysis without affecting the classical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent adipogenic pathways. Furthermore, our CD10-mediated screening analysis identified dexamethasone and retinoic acid as stimulator and inhibitor of adipogenesis, respectively, indicating CD10 as a useful biomarker for pro-adipogenic drug screening. Conclusion Overall, we establish CD10 as a functionally relevant ASC biomarker, which may be a prerequisite to identify high-quality cell populations for improving metabolic diseases.

scholarly journals Age-Related Yield of Adipose-Derived Stem Cells Bearing the Low-Affinity Nerve Growth Factor Receptor

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Raquel Cuevas-Diaz Duran ◽  
Maria Teresa González-Garza ◽  
Alejandro Cardenas-Lopez ◽  
Luis Chavez-Castilla ◽  
Delia Elva Cruz-Vega ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Growth Factor Receptor ◽  
Nerve Growth Factor Receptor ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Nerve Growth ◽  
Age Related

Adipose-derived stem cells (ADSCs) are a heterogeneous cell population that may be enriched by positive selection with antibodies against the low-affinity nerve growth factor receptor (LNGFR or CD271), yielding a selective cell universe with higher proliferation and differentiation potential. This paper addresses the need for determining the quantity of ADSCs positive for the CD271 receptor and its correlation with donor's age. Mononuclear cells were harvested from the lower backs of 35 female donors and purified using magnetic beads. Multipotency capacity was tested by the expression of stemness genes and through differentiation into preosteoblasts and adipocytes. A significant statistical difference was found in CD271+concentrations between defined age intervals. The highest yield was found within women on the 30–40-year-old age range. CD271+ADSCs from all age groups showed differentiation capabilities as well as expression of typical multipotent stem cell genes. Our data suggest that the amount of CD271+cells correlates inversely with age. However, the ability to obtain these cells was maintained through all age ranges with a yield higher than what has been reported from bone marrow. Our findings propose CD271+ADSCs as the primary choice for tissue regeneration and autologous stem cell therapies in older subjects.

scholarly journals Increase in Leptin and PPAR-γ Gene Expression in Lipedema Adipocytes Differentiated in vitro from Adipose-Derived Stem Cells

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 430 ◽  
Author(s):  
Sara Al-Ghadban ◽  
Zaidmara T. Diaz ◽  
Hallie J. Singer ◽  
Karya B. Mert ◽  
Bruce A. Bunnell
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Adipogenic Differentiation ◽  
Stromal Vascular Fraction ◽  
Connective Tissue Disorder ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Ppar Γ ◽  
Expression Of Genes

Lipedema is a painful loose connective tissue disorder characterized by a bilaterally symmetrical fat deposition in the lower extremities. The goal of this study was to characterize the adipose-derived stem cells (ASCs) of healthy and lipedema patients by the expression of stemness markers and the adipogenic and osteogenic differentiation potential. Forty patients, 20 healthy and 20 with lipedema, participated in this study. The stromal vascular fraction (SVF) was obtained from subcutaneous thigh (SVF-T) and abdomen (SVF-A) fat and plated for ASCs characterization. The data show a similar expression of mesenchymal markers, a significant increase in colonies (p < 0.05) and no change in the proliferation rate in ASCs isolated from the SVF-T or SVF-A of lipedema patients compared with healthy patients. The leptin gene expression was significantly increased in lipedema adipocytes differentiated from ASCs-T (p = 0.04) and the PPAR-γ expression was significantly increased in lipedema adipocytes differentiated from ASCs-A (p = 0.03) compared to the corresponding cells from healthy patients. No significant changes in the expression of genes associated with inflammation were detected in lipedema ASCs or differentiated adipocytes. These results suggest that lipedema ASCs isolated from SVF-T and SVF-A have a higher adipogenic differentiation potential compared to healthy ASCs.

scholarly journals Monocytes and pyrophosphate promote mesenchymal stem cell viability and early osteogenic differentiation

2022 ◽  
Vol 33 (1) ◽  
Author(s):  
Sara Svensson ◽  
Michael Palmer ◽  
Johan Svensson ◽  
Anna Johansson ◽  
Håkan Engqvist ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Culture Media ◽  
Conditioned Media ◽  
Human Mscs ◽  
Media Type ◽  
Inflammatory Monocytes ◽  
Ppar Γ

AbstractPyrophosphate-containing calcium phosphate implants promote osteoinduction and bone regeneration. The role of pyrophosphate for inflammatory cell-mesenchymal stem cell (MSC) cross-talk during osteogenesis is not known. In the present work, the effects of lipopolysaccharide (LPS) and pyrophosphate (PPi) on primary human monocytes and on osteogenic gene expression in human adipose-derived MSCs were evaluated in vitro, using conditioned media transfer as well as direct effect systems. Direct exposure to pyrophosphate increased nonadherent monocyte survival (by 120% without LPS and 235% with LPS) and MSC viability (LDH) (by 16–19% with and without LPS). Conditioned media from LPS-primed monocytes significantly upregulated osteogenic genes (ALP and RUNX2) and downregulated adipogenic (PPAR-γ) and chondrogenic (SOX9) genes in recipient MSCs. Moreover, the inclusion of PPi (250 μM) resulted in a 1.2- to 2-fold significant downregulation of SOX9 in the recipient MSCs, irrespective of LPS stimulation or culture media type. These results indicate that conditioned media from LPS-stimulated inflammatory monocytes potentiates the early MSCs commitment towards the osteogenic lineage and that direct pyrophosphate exposure to MSCs can promote their viability and reduce their chondrogenic gene expression. These results are the first to show that pyrophosphate can act as a survival factor for both human MSCs and primary monocytes and can influence the early MSC gene expression.

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.

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