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 ◽  
...  

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.

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.


2005 ◽  
Vol 17 (9) ◽  
pp. 67
Author(s):  
P. J. Verma ◽  
K. Upton ◽  
H. Mc Connell ◽  
I. Vassiliev

The isolation of stem cells has become an area of increasing interest due to their potential uses in animal reproduction, somatic cell nuclear transfer and cell therapies. The most attractive options are the isolation of stem cells from individual embryos or adult somatic tissues. In addition, for cell therapy, the use of autologous stem cells is considered to have an advantage over heterologous cell based therapies in that immune rejection issues would be circumvented. Here we report on our attempts to isolate stem cells from both these sources in a bovine model. Bovine ES-like (bES) cells were successfully isolated from embryos and maintained in vitro for up to six passages. These cells retained the morphology characteristic of bES cells: small cytoplasmic/nuclear ratio, nuclei with multiple nucleoli, and multiple lipid inclusions in cytoplasm. bES cell colonies grew as monolayers, as islands of ES cells surrounded by trophectoderm (TE) cells. Immunohistochemical detection of SSEA-1 and SSEA-4 demonstrated expression of these markers in bES cells but not in TE cells. Further, the expression of the pluripotent markers Oct-4, Rex-1 and SSEA-1 by RT-PCR was also detected in bES cells but not in TE cells. On spontaneous differentiation, these cells were able to form a variety of cell types including beating muscle with the cells displaying a propensity to differentiate in a manner reminiscent of human ES cells. (2) We also report the isolation of putative stem cells from adult bovine skin biopsies, which express the stem cell markers Oct-4 and SSEA-1 analysed by RT-PCR and are capable of forming 3-dimensional colonies. These cells are obtained from a skin biopsy, a relatively non-invasive technique that makes them useful as donors for therapeutic applications. In summary, we have identified populations of stem cells from embryonic and adult bovine tissues, which are readily isolated. Further characterization of the differentiation potential of these cells is needed to identify the suitability of this population for use in autologous stem cell therapies.


2010 ◽  
Vol 22 (1) ◽  
pp. 346 ◽  
Author(s):  
S. A. Choi ◽  
J. H. Lee ◽  
K. J. Kim ◽  
E. Y. Kim ◽  
X. X. Li ◽  
...  

The dog is biologically comparable with humans with respect to stem cell kinetics, haematopoietic demand, and responsiveness to cytokines. The availability of canine mesenchymal stem cells allows for the establishment of the dog as a large animal model for testing the safety and efficacy of mesenchymal stem cells replacement therapy. Large animal models, such as the dog, are invaluable for working out the practicalities of a therapeutic regimen in a complex system and for verifying established mechanistic theories. Therefore, canine stem cells present the potential for unique and exciting biological opportunities. Recent observations also indicate that stem cells derived from second-trimester amniocentesis are pluripotent, capable of differentiating into multiple lineages, including representatives of all 3 embryonic germ layers. Compared with embryonic stem cells, amniotic fluid stem cells can be obtained without destroying embryos, thus avoiding much ethical controversy. The aim of the current study was to investigate adipogenic, osteogenic, and chondrogenic in vitro differentiation potential of canine amniotic fluid-derived mesenchymal stem cells by biological characterization. We successfully isolated and characterized canine amniotic fluid-derived mesenchymal stem cells (cAFS). Expression of stem cell-specific marker OCT3/4, SOX2, and NANOG was confirmed by RT-PCR. Flow cytometric analysis showed that cAFS were positive for CD44, CD29, and CD90 but negative for CD34. Immunocytochemical analysis also showed the expression of alkaline phosphatase, SOX2, SSEA-1, and SSEA-4. Following incubation with specific adipogenic, osteogenic, and chondrogenic agents, cAFS stained positive by Oil Red O and Alizarin Red S, respectively. In conclusion, according to the preview studies on other mammalians, cAFS is an appropriate source of pluripotent stem cells. Here, we demonstrated that cAFS has a high adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Therefore, amniotic fluid might be a suitable alternative source of stem cells. This study was financially supported by KOSEF (grant #R01-2008-000-21076-0), research fund of Chungnam National University, and the Korean MEST, through the BK21 program for creative research in animal biotechnology.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2216-2216
Author(s):  
Chad Sanada ◽  
Evan J Colletti ◽  
Melisa Soland ◽  
Chung-Jung Kuo ◽  
Christopher D Porada ◽  
...  

Abstract Abstract 2216 The liver is considered to be the primary site of factor VIII (FVIII) production in the body; however, evidence is mounting that suggests there are secondary sites in which considerable synthesis of FVIII takes place. Studies of FVIII mRNA expression in various human tissues have revealed that FVIII message can be found throughout the body. Additionally, acute liver failure correlates with an increase in circulating FVIII levels. Some reports have identified endothelial cells as a significant extra-hepatic source of FVIII, possibly explaining both the widespread presence of FVIII mRNA and the increase in FVIII levels upon liver failure. However, the possibility exists that other cell types present throughout the body also produce FVIII and contribute to circulating FVIII levels. Mesenchymal Stem Cells (MSCs) represent a potential alternative; they are a diverse group of stromal cells which can be found in the perivascular regions of multiple tissues throughout the body. Previous studies demonstrated that MSCs are capable of efficiently producing and secreting high levels of FVIII in vitro when transduced with FVIII-encoding viral vectors, but to date, the innate ability of MSCs to produce FVIII has not been explored. As such, we investigated the potential for MSCs to produce endogenous FVIII message and secrete functional protein. MSCs isolated based on Stro-1 positivity from human lung, liver, brain, and bone marrow (BM) were grown in cell culture and assayed for production of FVIII message by both microarray analysis and RT-PCR. Microarray data showed that there were significant amounts of FVIII message in all four cell types tested and that the amount of message in BM MSCs was three-fold higher than each of the other three cell types. RT-PCR analysis confirmed the presence of FVIII message in all four MSC populations. Secretion of functional FVIII protein was subsequently measured using a chromogenic assay. MSC culture supernatants were collected for either 24 or 48 hours, and FVIII activity was determined using pooled normal human plasma as a control to create a standard curve. FVIII activity in the supernatants of MSCs was in the range of 0.6 to 2.0 mU/1×10^6 cells/ 24hr. Moreover, MSCs continued to express and produce FVIII during time in culture until our last evaluation at passage 20, indicating that there is an innate ability of these cells to continually produce FVIII. Taken together, these data demonstrate that human MSCs are capable of producing and secreting functional FVIII in vitro, and given their widespread location throughout the body, this finding raises the possibility that, in vivo, these cells might significantly contribute to the total FVIII pool. This is the first report, to our knowledge, that implicates MSCs as a potential endogenous source for circulating FVIII. Further studies of in vivo FVIII expression by MSCs are warranted and may provide a clearer understanding of extra-hepatic FVIII production in the body while aiding in the discovery of novel therapies for hemophilia A. Disclosures: No relevant conflicts of interest to declare.


2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Lucas-Sebastian Spitzhorn ◽  
Md Shaifur Rahman ◽  
Laura Schwindt ◽  
Huyen-Tran Ho ◽  
Wasco Wruck ◽  
...  

Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology,in vitrodifferentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFβ-signalling. An AF-MSC-enriched gene set included putative AF-MSC markersPSG5,EMX-2, andEVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling.


2019 ◽  
Vol 7 (4) ◽  
pp. 507-515 ◽  
Author(s):  
Eman E. A. Mohammed ◽  
Mohamed El-Zawahry ◽  
Abdel Razik H. Farrag ◽  
Nahla N. Abdel Aziz ◽  
Wessam Sharaf-ElDin ◽  
...  

BACKGROUND: Cell therapies offer a promising potential in promoting bone regeneration. Stem cell therapy presents attractive care modality in treating degenerative conditions or tissue injuries. The rationale behind this is both the expansion potential of stem cells into a large cell population size and its differentiation abilities into a wide variety of tissue types, when given the proper stimuli. A progenitor stem cell is a promising source of cell therapy in regenerative medicine and bone tissue engineering. AIM: This study aimed to compare the osteogenic differentiation and regenerative potentials of human mesenchymal stem cells derived from human bone marrow (hBM-MSCs) or amniotic fluid (hAF-MSCs), both in vitro and in vivo studies. SUBJECTS AND METHODS: Human MSCs, used in this study, were successfully isolated from two human sources; the bone marrow (BM) and amniotic fluid (AF) collected at the gestational ages of second or third trimesters. RESULTS: The stem cells derived from amniotic fluid seemed to be the most promising type of progenitor cells for clinical applications. In a pre-clinical experiment, attempting to explore the therapeutic application of MSCs in bone regeneration, Rat lumbar spines defects were surgically created and treated with undifferentiated and osteogenically differentiated MSCs, derived from BM and second trimester AF. Cells were loaded on gel-foam scaffolds, inserted and fixed in the area of the surgical defect. X-Ray radiography follows up, and histopathological analysis was done three-four months post- operation. The transplantation of AF-MSCs or BM-MSCs into induced bony defects showed promising results. The AF-MSCs are offering a better healing effect increasing the likelihood of achieving successful spinal fusion. Some bone changes were observed in rats transplanted with osteoblasts differentiated cells but not in rats transplanted with undifferentiated MSCs. Longer observational periods are required to evaluate a true bone formation. The findings of this study suggested that the different sources; hBM-MSCs or hAF-MSCs exhibited remarkably different signature regarding the cell morphology, proliferation capacity and osteogenic differentiation potential CONCLUSIONS: AF-MSCs have a better performance in vivo bone healing than that of BM-MSCs. Hence, AF derived MSCs is highly recommended as an alternative source to BM-MSCs in bone regeneration and spine fusion surgeries. Moreover, the usage of gel-foam as a scaffold proved as an efficient cell carrier that showed bio-compatibility with cells, bio-degradability and osteoinductivity in vivo.


2011 ◽  
Vol 23 (1) ◽  
pp. 252 ◽  
Author(s):  
L. Valentini ◽  
M. Filioli Uranio ◽  
A. Lange Consiglio ◽  
A. C. Guaricci ◽  
M. Caira ◽  
...  

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).


2019 ◽  
Vol 14 (4) ◽  
pp. 327-336 ◽  
Author(s):  
Carl R. Harrell ◽  
Marina Gazdic ◽  
Crissy Fellabaum ◽  
Nemanja Jovicic ◽  
Valentin Djonov ◽  
...  

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.


2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Chad M. Teven ◽  
Xing Liu ◽  
Ning Hu ◽  
Ni Tang ◽  
Stephanie H. Kim ◽  
...  

Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES) cells. Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.


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