312 ISOLATION, PROLIFERATION, AND CHARACTERIZATION OF MESENCHYMAL STEM CELLS FROM AMNIOTIC FLUID, AMNION, AND UMBILICAL CORD MATRIX IN THE DOG

2011 ◽  
Vol 23 (1) ◽  
pp. 252 ◽  
Author(s):  
L. Valentini ◽  
M. Filioli Uranio ◽  
A. Lange Consiglio ◽  
A. C. Guaricci ◽  
M. Caira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Fluid ◽  
Cell Viability ◽  
Umbilical Cord ◽  
Cell Biology ◽  
Cell Types ◽  
Telomerase Activity ◽  
Umbilical Cord Matrix

Mesenchymal stem cells (MSC) are defined as multipotent stem cells that can differentiate into various cell types in vivo and in vitro under controlled conditions. These cells express specific markers detectable by analysis at the mRNA or protein level. Important sources of MSC could be fetal adnexa, such as amniotic fluid (AF), amnion (AM), and umbilical cord matrix (UCM). Canine MSC should be of use for cell-based therapies and tissue engineering improving treatment of several diseases. Moreover, the dog has been considered an attractive animal model to study human diseases. In the present study, we successfully isolated and molecularly characterised AF-MSC, AM-MSC, and UCM-MSC from dogs. Chromosomal stability and telomerase activity were also investigated. Samples were recovered after elective ovariohysterectomy in 3 bitches 25 to 40 days of gestational age. After isolation, cells were maintained in culture (Bossolasco et al. 2006 Cell Res. 16, 329–336) for different passages to perform growth and doubling time (DT) studies. Expression analyses of embryonic (Oct-4, Nanog), mesenchymal (CD44, CD184, CD29), and haematopoietic (CD34, CD45) markers were carried out by RT-PCR. Karyotype analysis was performed by Q banding. Telomerase activity was analysed by TRAPeze Telomerase Detection Kit. In all 3 cell types, the morphology of proliferating cells appeared typically fibroblast-like. In the growth study, cells isolated from AF and AM were cultured until P3, and cells isolated from UCM were maintained until P7. The population DT in AF-MSC was significantly increased (Student’s t-test: P < 0.05) when comparing P1 v. P4. In AM-MSC, DT increased significantly in P1 v. P2 (P < 0.001), and in UCM-MSC, DT significantly increased in P1 v. P4 (P < 0.001). In AF-MSC, cell viability did not change with passages. In AM-MSC, cell viability significantly decreased (P < 0.001) between P1 and P4. In UCM-MSC, cell viability remained at approximately constant levels up to P6 and significantly decreased at P7 (P < 0.001). Amnion and UCM-MSC expressed Oct-4 and CD44, CD184, and CD29, whereas AF-MSC expressed only Oct-4 and CD44. Nanog, CD34, and CD45 were never found to be expressed in any cell line at any passage. In all cell lines, analysed metaphases at P4 showed normal chromosomal number and structure. Telomerase activity was observed in UCM-MSC, whereas tests on AF and AM-MSC are still on going. We first reported data on isolation, in vitro culture, and characterisation of MSC from AM and UCM in the dog. Cells expressed embryonic and MSC markers beginning at P1 and showed normal karyotype. These data indicated that canine MSC from fetal adnexa could be used to study stem cell biology and their application in therapeutic programs. Financial support was provided by Fondi di Ateneo 2009. University of Bari Aldo Moro (COD. ORBA09UDWX) (Resp. Sci. Maria Elena Dell’Aquila).

scholarly journals Effects on Proliferation and Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells Engineered to Express Neurotrophic Factors

2016 ◽  
Vol 2016 ◽  
pp. 1-11
Author(s):  
Yi Wang ◽  
Youguo Ying ◽  
Xiaoyan Cui
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Neurotrophic Factors ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Human Umbilical Cord ◽  
Proliferation And Differentiation

Mesenchymal stem cells (MSCs) are multipotential cells with capability to form coloniesin vitroand differentiate into distinctive end-stage cell types. Although MSCs secrete many cytokines, the efficacy can be improved through combination with neurotrophic factors (NTFs). Moreover, MSCs are excellent opportunities for local delivery of NTFs into injured tissues. The aim of this present study is to evaluate the effects of overexpressing NTFs on proliferation and differentiation of human umbilical cord-derived mesenchymal stem cells (HUMSCs). Overexpressing NTFs had no effect on cell proliferation. Overexpressing NT-3, BDNF, and NGF also had no significant effect on the differentiation of HUMSCs. Overexpressing NTFs all promoted the neurite outgrowth of embryonic chick E9 dorsal root ganglion (DRG). The gene expression profiles of the control and NT-3- and BDNF-modified HUMSCs were compared using RNA sequencing and biological processes and activities were revealed. This study provides novel information about the effects of overexpressing NTFs on HUMSCs and insight into the choice of optimal NTFs for combined cell and gene therapy.

scholarly journals Human Amniotic Fluid Stem Cells: General Characteristics and Potential Therapeutic Applications

2021 ◽  
Author(s):  
Seyed Mehdi Hoseini ◽  
Maryam Moghaddam-Matin ◽  
Ahmad Reza Bahrami ◽  
Fateme Montazeri ◽  
Seyed Mehdi Kalantar
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Regenerative Medicine ◽  
Amniotic Fluid ◽  
Adult Stem Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Telomerase Activity ◽  
Clinical Applications ◽  
Embryoid Bodies

Introduction: Amniotic fluid contains a mixture of different cell types sloughed from the fetal skin, respiratory, alimentary and urogenital tracts, as well as the amnion membrane. As amniotic fluid develops prior to the process of gastrulation, many cells found in its heterogeneous population do not undergo lineage specialization. Therefore, amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may correspond to a new class of stem cells with properties of intermediate plasticity between pluripotent and adult stem cell types. Compared to mesenchymal stem cells (MSCs) from other sources, such as bone marrow, AF-MSCs have better properties for clinical applications, such as differentiation into the cells of three germ layers, high clonal capacity, ability to form embryoid bodies, expression of pluripotent markers, high self-renewal capacity (over 250 population doublings) with normal karyotype at late passages, long telomere length due to continued telomerase activity, specially non-tumorigenicity, low immunogenicity, anti-inflammatory and immunomodulatory properties. Conclusion: Such features have nominated AF-MSC for a range of clinical applications, including in regenerative medicine. In several studies, these cells have been used to regenerate nerve, lung, and heart tissues. Overall, AF-MSCs are expected to be an ideal source of stem cells for future regenerative medicine and tissue engineering.

scholarly journals Insulin Promotes the Proliferation of Human Umbilical Cord Matrix-Derived Mesenchymal Stem Cells by Activating the Akt-Cyclin D1 Axis

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Peng Li ◽  
Jinsong Wei ◽  
Xiang Gao ◽  
Bo Wei ◽  
Hao Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cyclin D1 ◽  
Umbilical Cord ◽  
Cell Cycle Progression ◽  
Human Umbilical Cord ◽  
Expression Levels ◽  
Umbilical Cord Matrix ◽  
Phosphorylated Akt

Background. The functions of insulin in mesenchymal stem cells (MSC) remain poorly understood. Methods. MSC from human umbilical cord matrix (UCM) cultured in serum-free media (SFM) with or without insulin were subjected to various molecular biological analyses to determine their proliferation and growth states, expression levels of Akt-cyclin D1 signaling molecules, and in vitro differentiation capacities. Results. Insulin accelerated the G1-S cell cycle progression of UCM-MSC and significantly stimulated their proliferation and growth in SFM. The pro-proliferative action of insulin was associated with augmented cyclin D1 and phosphorylated Akt expression levels. Akt inactivation remarkably abrogated insulin-induced increases in cyclin D1 expression and cell proliferation, indicating that insulin enhances the proliferation of UCM-MSC via acceleration of the G1-S transition mediated by the Akt-cyclin D1 pathway. Additionally, the UCM-MSC propagated in SFM supplemented with insulin exhibited similar specific surface antigen profiles and differentiation capacities as those generated in conventional media containing fetal bovine serum. Conclusions. These findings suggest that insulin acts solely to promote UCM-MSC proliferation without affecting their immunophenotype and differentiation potentials and thus have important implications for utilizing insulin to expand clinical-grade MSC in vitro.

186 BOVINE AMNIOTIC FLUID MESENCHYMAL STEM CELLS CHARACTERIZATION AFTER CULTURE IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 207
Author(s):  
B. Rossi ◽  
B. Merlo ◽  
E. Iacono ◽  
P. P. Pagliaro ◽  
P. L. Tazzari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Fluid ◽  
Cell Types ◽  
Calcium Deposition ◽  
Cell Populations ◽  
Rt Pcr ◽  
Differentiation Potential ◽  
Phenotypic Characterisation

In recent years, fetal adnexa and fluids have been recognised as important sources of mesenchymal stem cells (MSC). The aim of this study was to characterise cell populations of bovine amniotic fluid, studying phenotypic characterisation, RNA expression, and differentiation potential of samples after in vitro culture for different lengths of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at the slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1 : 1) plus 10% fetal bovine serum (FBS) in 5% CO2 at 38.5°C. At passages P3 and P7, a sample for each of the 4 population found was characterised. Immunophenotypic characterisation was performed for MSC (CD90, CD105, CD44) and haematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4, and α-SMA and the ratio between positive cells and total nuclei was evaluated. Gene expression profile was analysed by RT–PCR for pluripotency markers (Oct4, Nanog, Sox2). At the same passages chondrogenic, osteogenic and adipogenic differentiation were induced and evaluated morphologically and cytologically using, respectively, Alcian blue to identify cartilage matrix, Von Kossa for extracellular calcium deposition, and Oil Red O for intracellular lipid droplets. Cell population appeared heterogeneous and we could identify 2 main cell types: round (R) and spindle-shaped (S) cells. Each isolated sample was classified into one of the following 4 types depending on percentages of R or S cells: prevalence of S-cells (S), prevalence of R-cells (R), and samples showing both morphologies with ~10% of S-cells (S10) or 40% S-cells (S40). S-cells percentage decreased with passages in S10 and S40. After FACS, all lines were positive for CD90, CD105, CD44, and CD34 and negative for CD14 both at P3 and at P7. After ICC, Oct4 was negative in all samples analysed, few S cells stained for SSEA4 (8%) at P3 but increased at P7 to 22%; R, S10, and S40 did not express SSEA4 both at P3 and at P7. α-SMA was expressed in all samples at P3 (9.4% S; 0.9% R; 2.5% S10; 27% S40) but not at P7 (27.5% S; 0% R; 0% S10; 0% S40). After RT–PCR analyses, Oct4 was negative in all samples; at P3, Nanog was clearly positive in S-cells, weak in S40, and negative in R and S10, but all samples turned negative at P7. Sox2 was weakly expressed (S) or not expressed (S10, S40, R) at P3 and it was negative in all cells at P7. Only S showed high differentiation potential into all 3 lineages at both P3 and P7, R had the lowest differentiation potential, whereas S10 and S40 were intermediate at both end points. In conclusion, bovine amniotic fluid showed heterogeneous cell populations and S-type had the characteristics of MSCs. S10 and S40 showed more MSC markers at P3, when S cells were still present, and this aspect suggests that S population is the presumptive MSC one. Although prevalent, R-type showed only some MSC characteristics. Further studies are under way to improve S-type isolation, purification, and culture, and to determine the lifespan of these cell types. This work was supported by grant PRIN2009.

Immunosuppressive function of mesenchymal stem cells from human umbilical cord matrix in immune thrombocytopenia patients

2012 ◽  
Vol 107 (05) ◽  
pp. 937-950 ◽  
Author(s):  
Li Ma ◽  
Zeping Zhou ◽  
Donglei Zhang ◽  
Shaoguang Yang ◽  
Jinhong Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Immune Thrombocytopenia ◽  
Mononuclear Cells ◽  
Human Umbilical Cord ◽  
Therapeutic Effects ◽  
Peripheral Blood Mononuclear ◽  
Umbilical Cord Matrix

SummaryHuman umbilical cord matrix/Wharton's Jelly (hUC)-derived mesenchymal stem cells (MSC) have been shown to have marked therapeutic effects in a number of inflammatory diseases and autoimmune diseases in humans based on their potential for immunosuppression and their low immunogenicity. Currently, no data are available on the effectiveness of UC-MSC transplantation in immune thrombocytopenia (ITP) patients. It was the objective of this study to assess the effect of allogeneic UC-MSCs on ITP patients in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BM-MNCs) from ITP patients and healthy controls were co-cultured with UC-MSCs for three days and seven days, respectively. Flow cytometry and ELISA were applied to assess the various parameters. In PBMCs from ITP patients, the proliferation of autoreactive T, B lymphocytes and destruction of autologous platelets were dramatically suppressed by UC-MSCs. UC-MSCs not only suppressed co-stimulatory molecules CD80, CD40L and FasL expression but also in shifting Th1/Th2/Treg cytokines profile in ITP patients. UC-MSCs obviously reversed the dysfunctions of megakaryocytes by promoting platelet production and decreasing the number of living megakaryocytes as well as early apoptosis. In addition, the level of thrombopoietin was increased significantly. Our clinical study showed that UC-MSCs play a role in alleviating refractory ITP by increasing platelet numbers. These findings suggested that UC-MSCs transplantation might be a potential therapy for ITP.

Ochratoxin A at low concentrations inhibits in vitro growth of canine umbilical cord matrix mesenchymal stem cells through oxidative chromatin and DNA damage

2015 ◽  
Vol 57 ◽  
pp. 121-129 ◽  
Author(s):  
Lucia Rutigliano ◽  
Luisa Valentini ◽  
Nicola Antonio Martino ◽  
Flavia Pizzi ◽  
Antonina Zanghì ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Mesenchymal Stem Cells ◽  
Ochratoxin A ◽  
Umbilical Cord ◽  
In Vitro Growth ◽  
Low Concentrations ◽  
Umbilical Cord Matrix

scholarly journals Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Stimulated by Hypoxia Promote Angiogenesis Both In Vitro and In Vivo

2012 ◽  
Vol 21 (18) ◽  
pp. 3289-3297 ◽  
Author(s):  
Hong-Chao Zhang ◽  
Xin-Bin Liu ◽  
Shu Huang ◽  
Xiao-Yun Bi ◽  
Heng-Xiang Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Human Umbilical Cord

Epigenetic features in regulation of telomeres and telomerase in stem cells

2021 ◽  
Author(s):  
Fatma Dogan ◽  
Nicholas R. Forsyth
Keyword(s):  
Stem Cells ◽  
Telomere Length ◽  
Cell Biology ◽  
Replicative Senescence ◽  
Telomerase Activity ◽  
Cell Phenotype ◽  
Tissue Degeneration ◽  
Unique System ◽  
Telomerase Regulation

The epigenetic nature of telomeres is still controversial and different human cell lines might show diverse histone marks at telomeres. Epigenetic modifications regulate telomere length and telomerase activity that influence telomere structure and maintenance. Telomerase is responsible for telomere elongation and maintenance and is minimally composed of the catalytic protein component, telomerase reverse transcriptase (TERT) and template forming RNA component, telomerase RNA (TERC). TERT promoter mutations may underpin some telomerase activation but regulation of the gene is not completely understood due to the complex interplay of epigenetic, transcriptional, and posttranscriptional modifications. Pluripotent stem cells (PSCs) can maintain an indefinite, immortal, proliferation potential through their endogenous telomerase activity, maintenance of telomere length, and a bypass of replicative senescence in vitro. Differentiation of PSCs results in silencing of the TERT gene and an overall reversion to a mortal, somatic cell phenotype. The precise mechanisms for this controlled transcriptional silencing are complex. Promoter methylation has been suggested to be associated with epigenetic control of telomerase regulation which presents an important prospect for understanding cancer and stem cell biology. Control of down-regulation of telomerase during differentiation of PSCs provides a convenient model for the study of its endogenous regulation. Telomerase reactivation has the potential to reverse tissue degeneration, drive repair, and form a component of future tissue engineering strategies. Taken together it becomes clear that PSCs provide a unique system to understand telomerase regulation fully and drive this knowledge forward into aging and therapeutic application.

scholarly journals Amniotic fluid stem cells ameliorate cisplatin-induced acute renal failure through induction of autophagy and inhibition of apoptosis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ekta Minocha ◽  
Rohit Anthony Sinha ◽  
Manali Jain ◽  
Chandra Prakash Chaturvedi ◽  
Soniya Nityanand
Keyword(s):  
Stem Cells ◽  
Renal Failure ◽  
Acute Renal Failure ◽  
Amniotic Fluid ◽  
Cell Types ◽  
Treated Group ◽  
Protective Role ◽  
Protective Effects ◽  
Amniotic Fluid Stem Cells

Abstract Background We have recently demonstrated that amniotic fluid stem cells (AFSC) express renal progenitor markers and can be differentiated in vitro into renal lineage cell types, viz, juxtaglomerular and renal proximal tubular epithelial-like cells. Here, we have evaluated the therapeutic efficacy of AFSC in a cisplatin-induced rat model of acute renal failure (ARF) and investigated the underlying mechanisms responsible for their renoprotective effects. Methods ARF was induced in Wistar rats by intra-peritoneal injection of cisplatin (7 mg/kg). Five days after cisplatin injection, rats were randomized into two groups and injected with either AFSC or normal saline intravenously. On days 8 and 12 after cisplatin injection, the blood biochemical parameters, histopathological changes, apoptosis and expression of pro-apoptotic, anti-apoptotic, and autophagy-related proteins in renal tissues were studied in both groups of rats. To further confirm whether the protective effects of AFSC on cisplatin-induced apoptosis were dependent on autophagy, chloroquine, an autophagy inhibitor, was administered by the intra-peritoneal route. Results Administration of AFSC in ARF rats resulted in improvement of renal function and attenuation of renal damage as reflected by significant decrease in blood urea nitrogen, serum creatinine levels, tubular cell apoptosis as assessed by Bax/Bcl2 ratio, and expression of the pro-apoptotic proteins, viz, PUMA, Bax, cleaved caspase-3, and cleaved caspase-9, as compared to the saline-treated group. Furthermore, in the AFSC-treated group as compared to the saline-treated group, there was a significant increase in the activation of autophagy as evident by increased expression of LC3-II, ATG5, ATG7, Beclin1, and phospho-AMPK levels with a concomitant decrease in phospho-p70S6K and p62 expression levels. Chloroquine administration led to significant reduction in the anti-apoptotic effects of the AFSC therapy and further deterioration in the renal structure and function caused by cisplatin. Conclusion AFSC led to amelioration of cisplatin-induced ARF which was mediated by inhibition of apoptosis and activation of autophagy. The protective effects of AFSC were blunted by chloroquine, an inhibitor of autophagy, highlighting that activation of autophagy is an important mechanism of action for the protective role of AFSC in cisplatin-induced renal injury.

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