Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

Laboratory of Animal Virology and Cell Culture, UNESP - University of São Paulo State, Araçatuba, São Paulo , 16050-680, Brazil. .
BMC Biotechnology (Impact Factor: 2.03). 05/2012; 12(1):18. DOI: 10.1186/1472-6750-12-18
Source: PubMed


The possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton's jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking.
Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells was included in the culture which demonstrated the immunossupression profile typically observed among isolated mesenchymal cells from other species. After classified the UC-WJ cells as mesenchymal stromal phenotype the in vitro 3D cultures was performed using the AlgiMatrix® protocol. Based on the size of spheroids (283,07 μm ± 43,10 μm) we found that three weeks of culture was the best period to growth the UC-WJ cells on 3D dimension. The initial cell density was measured and the best value was 1.5 × 106 cells/well.
We described for the first time the isolation and characterization of UC-WJ cells in a serum-free condition and maintenance of primitive mesenchymal phenotype. The culture was stable under 60 consecutive passages with no genetic abnormalities and proliferating ratios. Taken together all results, it was possible to demonstrate an easy way to isolate and culture of bovine-derived UC-WJ cells under 2D and 3D serum-free condition, from fetal adnexa with a great potential in cell therapy and biotechnology.

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    • "Little information is available regarding expression of MSC surface markers in bMSC. Analyses by flow cytometry in bMSC isolated from the Wharton’s jelly detected high proportion of cells positive for CD29, CD73, CD90 and CD105 and negative for CD34 and CD45 [11]. Using Q-PCR, we reported a similar MSC marker profile in bMSC isolated from BM of abattoir-derived bovine fetuses [12]. "
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    ABSTRACT: Background Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses. Results Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes. Conclusion The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine.
    BMC Veterinary Research 07/2014; 10(1):154. DOI:10.1186/1746-6148-10-154 · 1.78 Impact Factor
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    • "It is now evident that the secretions from MSCs have potent angiogenic, anti-apoptotic and anti-scarring properties (Rehman et al. 2004) which, along with their ease of administration , make them a powerful potential therapy for veterinary use. As of now limited reports are available regarding the isolation and characterization of whartons jelly derived MSCs in farm animal species (Singh et al. 2013; Cardoso et al. 2012; Seo et al. 2012). In caprine, the preferred ruminant model for biological experimentations and regenerative therapy, isolation and characterization of WJ-MSCs were first reported by Babaei et al. (2008) and later by Azari et al. (2011). "
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    ABSTRACT: Aim of the present study was in vitro expansion and characterization of caprine wharton's jelly derived mesenchymal stem cells (cWJ-MSCs) to investigate their tissue healing potential in xenogenic animal model. Plastic adherent fibroblastoid cell populations with distinctive homogeneous morphology were isolated from caprine wharton's jelly explants. These wharton's jelly derived cells were found positive for the surface markers CD-73, STRO-1 and CD-105, whereas they were negative for hematopoetic stem cell marker CD-34. In vitro cultured cWJ-MSCs also showed differentiation properties into osteogenic, adipogenic and chondrogenic lineages as demonstrated by von Kossa, Oil Red- O and Alcian blue staining respectively, which was further confirmed and quantified by flow cytometric analysis. Furthermore, these well characterized cWJ-MSCs were evaluated for the wound-healing potential in full-thickness skin wounds in rabbit model for 28 days. Caprine WJ- MSCs treated skin wounds showed significantly (P < 0.05) higher percentage of wound contraction especially at the 21(st) day post transplantation when compared to PBS treated control group animals. Further, we observed better healing potential of cWJ-MSCs in terms of histo-morphological evaluation, epithelialisation and collagenization with matured vascularization stage by day 28 as compared to control. In conclusion, cWJ- MSCs provide an alternative inexhaustible source of mesenchymal stem cells and also unravel new perspectives pertaining to the therapeutic use of these cells in different species.
    Veterinary Research Communications 03/2014; 38(2). DOI:10.1007/s11259-014-9597-y · 1.24 Impact Factor
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    • "Mesenchymal stem cells (MSCs) are multipotent progenitor cells and they can be cultured from adult and fetal tissues (Cremonesi et al., 2011). Recently, a bovine mesenchymal cell line has been described, where it was isolated from Wharton's jelly layer (Cardoso et al., 2012). This biological characteristic of immune regulation has been demonstrated by others (Cremonesi et al., 2011). "
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    ABSTRACT: Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny.
    Genetics and molecular research: GMR 10/2013; 12(3):3897-3904. DOI:10.4238/2013.September.23.8 · 0.78 Impact Factor
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