Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: A comparative study of amniotic fluid, amnion, and umbilical cord matrix

Abstract

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

Amniotic Fluid-Derived Stem Cells for Cardiovascular Tissue Engineering Applications

“…19,[23][24][25][26][27][28] Additionally, some researchers are comparing AFSC to other fetal-derived tissues such as umbilical cord blood and amniotic membrane. 27,29,30 AFSC are being explored for use in tissue engineering applications for a variety of tissue types. This review focuses on cardiovascular cell types and tissue engineering, which are addressed further below, but AFSC are also being investigated for osteogenic, 13,31,32 renal, 33,34 myogenic, 35 epithelial, 36,37 and neuronal 14,[38][39][40][41][42][43][44][45] differentiation and tissue engineering.…”

“…18 Oct4, Sox2, and Nanog, transcription factors necessary for maintaining pluirpotency in ESCs, also displayed mixed results across different studies of AFSC. 16,17,20,23,24,[29][30][31]35,37,[39][40][41]46,[48][49][50][51][52]54 In Vitro Cardiac Myocyte Differentiation of AFSC AFSC have been investigated for cardiac tissue engineering. 55 AFSC applications to in vitro cardiac tissue engineering are summarized in this section and Table 3.…”

Calcium-Sensing Receptor-Mediated Osteogenic and Early-Stage Neurogenic Differentiation in Umbilical Cord Matrix Mesenchymal Stem Cells from a Large Animal Model

“…Osteogenic and neurogenic in vitro differentiation potential was induced as previously reported [21], [50]. Cells at P3 were seeded at a density of 3000 cells /cm 2 in six-well plates and were cultured until they reached approximately 80–90% confluence.…”

Effect of Passage, Sorting, and Media on Differentiation Capacity and Marker Expression in Amniotic Fluid Stem Cells

“…[3][4][5][6][7][8]10,11,14,15,25,26,28,29 However, other studies have used AFSC maintained in medium containing 10% FBS 1,2,18,24 or 20% FBS. 16,22,30,34,35,37 In addition to culture of AFSC in medium supplemented with Chang medium, studies have also cultured AFSC in medium with no additional supplements, 1,16,24 5-10 ng/ml basic fibroblast growth factor (bFGF), 17,[19][20][21]31 10 ng/ml epidermal growth factor (EGF), 13,18 or 10 ng/ml each bFGF and EGF. 36,37 To address the maintenance of stem cell markers and differentiation capacity of AFSC isolated and cultured in different conditions, both c-kit sorted and unfractionated AFSC from the same patient samples were cultured in a variety of medium conditions, including varying FBS concentration from 10 to 20% and testing different medium supplements.…”