Mesenchymal progenitor cells in the human umbilical cord
ABSTRACT Mesenchymal progenitor or stem cells (MPCs) isolated from fetal blood, liver, and bone marrow are a population of multipotential cells that can proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat, and stroma. The objective of this study was to isolate and characterize MPCs in the human umbilical cord. The suspensions of endothelial and subendothelial cells in cord vein were collected and cultured in M199 supplemented with 10% fetal bovine serum (FBS). Of 50 umbilical cord samples, 3 had numerous fibroblastoid cells morphologically distinguishable from endothelial cells. Fibroblastic cells displayed lack of expression of vWF, Flk-1, and PECAM-1, indicating the endothelial cell-specific marker. To investigate the differentiation potentials, the cells were cultured in adipogenic or osteogenic medium for 2 weeks. Fibroblast-like cells treated with adipogenic supplementation showed Oil red O-positive staining and expressed adipsin, FABP4, LPL, and PPARgamma2 genes by reverse transcriptase polymerase chain reaction (RT-PCR). In osteogenic differentiation, alkaline phosphatase activity and calcium accumulation were detected. RT-PCR studies determined that Cx43, osteopontin, and Runx2 genes were expressed in the osteogenic cultures. Among three cell lines cultured continuously for passage 10, two had normal karyotypes; however, one retained a karyotype of mos 46,XY/47,XY,+mar. These observations suggest that MPCs are present in human umbilical cord and possess several typical traits of MPCs.
- SourceAvailable from: Laura Elizabeth Sidney
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- "First was a standard keratocyte medium (KM), [17,46] consisting of DMEM (Gibco, Invitrogen, Paisley, UK), supplemented with 10% vol/vol heat-inactivated FBS (Fisher Scientific), 0.02 μg/ml gentamicin, 0.5 ng/ml amphotericin B (Gibco), 4.5 μg/ml insulin, human recombinant (Gibco), and 0.5% vol/vol DMSO (Sigma Aldrich). The second was a medium previously shown to support the expansion of MSCs [15,61-63] (MM), consisting of M199 medium (Sigma) supplemented with 20% vol/vol heat-inactivated FBS, 2.5 μg/ml antibiotic solution, Plasmocin (Autogen Bioclear, Wiltshire, UK), 0.02 μg/ml gentamicin, 0.5 ng/ml amphotericin B (Gibco), and 1.59 mM L-glutamine (Sigma Aldrich). "
ABSTRACT: The corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSC) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor has been hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability. PLCSC were grown in traditional Dulbecco's modified Eagle's medium (DMEM)-based keratocyte culture medium and an M199-based medium and analysed for a profile of cell surface markers using flow cytometry and differentiated into mesenchymal phenotypes analysed by qPCR and histological staining. PLCSC in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression using fluorescent activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSC and subpopulations were also cultured for epithelial transdifferentiation. Cells cultured in M199 demonstrated a more stem-like cell surface marker profile and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSC could be divided into CD34+CD105+, CD34-CD105+ and CD34-CD105- subpopulations, of which CD34+CD105+ cells were the most stem-like with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSC exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34+CD105+ showed greatest differentiation potential producing CK3+ and CK19+ cells, and expressed a range of both epithelial progenitor (HES1, FRZB1, DCT, SOD2, ABCG2, CDH1, KRT19) and terminally differentiated (DSG3, KRT3, KRT12, KRT24) genes. Culture medium has a significant effect on the phenotype and differentiation capacity of PLCSC. The stroma contains a heterogeneous cell population in which we have identified CD34+ cells as a stem cell population with a capacity for mesenchymal and epithelial differentiation.Stem Cell Research & Therapy 06/2013; 4(3):75. DOI:10.1186/scrt226 · 4.63 Impact Factor
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- "UCMSCs isolated from cord tissue samples processed after 04 days and 30 days of collection were incubated with growth medium containing 0.25mg of colcemid. After 4 hours of incubation the cells were harvested and resuspended in 0.075M KCl and then fixed in 3:1 methanol/acetic acid.11 GTG banding was done on metaphase spreads obtained from cultured UCMSCs. "
ABSTRACT: Umbilical cord tissue is a very rich source of mesenchymal stem cells. Instead of discarding this source we are banking the tissue along with cord blood for possible future cell based applications. The cord tissue needs to be transported and stored properly in order for it to be good enough for cell isolation at a later date. In this paper we have carried out a validation study to determine the maximum permissible time between cord tissue collection and beginning of cell culture process under defined conditions of temperature and collection media. Ten cord tissue samples were used for this study. The umbilical cord tissue segments were transported and stored at 2 - 8°(C) for varying periods of time viz. 04, 11, 22 and 30 days in a defined medium after which MSCs were isolated and characterized by flow cytometry. Karyotypic studies were also performed on the isolated cells at the above time points. MSCs could be successfully isolated from 09 even samples after a storage period of 22 days and from 07 samples after a period of 30 days from the date of collection. There was no change in the morphology, immunophenotye, karyotypye and growth potential of the cells isolated from cord tissue after the maximum storage period of 30 days. The umbilical cord tissue is stable for as long as 22 days if stored at the recommended storage conditions of 2 - 8°(C) in the defined medium.03/2013; 7(4):15-23.
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- "Human umbilical cord blood cells (HUCBCs) are a source of hematopoietic stem cells, endothelial cell precursors, mesenchymal progenitors, and other multipotent/ pluripotent lineage stem cells and represent a promising option for alternative for experimental stroke therapies (Chen et al., 2001, 2006; Kim et al., 2004; Vendrame et al., 2004, 2005; Boltze et al., 2005, 2011b; Newman et al., 2005; Berger et al., 2006; Newcomb et al., 2006; Bewley and Mercer, 2010). One of the contributing factors for their therapeutic efficacy for experimental stroke is that HUCBCs provide a ready supply of neurotrophic and angiogenic factors, and induce neurogenesis and angiogenesis (Chen et al., 2001, 2007; Hau et al., 2008; Jiang et al., 2008; Chung et al., 2009; Liu et al., 2009; Park et al., 2009; Arien-Zakay et al., 2011; Terry et al., 2011; Nih et al., 2012). "
ABSTRACT: Human umbilical cord blood cells (HUCBCs) have been employed as a restorative treatment for experimental stroke. In this study, we investigated whether transplantation of sub-therapeutic doses of HUCBCs and Simvastatin enhances cerebral vascular remodeling after stroke. Adult male Wistar rats (n=34) were subjected to transient middle cerebral artery occlusion (MCAo) and treated with: phosphate-buffered solution (PBS, gavaged daily for 7days); Simvastatin (0.5mg/kg, gavaged daily for 7days); HUCBCs (1×10(6), injected once via tail vein); and combination Simvasatin with HUCBCs, starting at 24h after MCAo. There was no significant difference between Simvastatin- or HUCBC-monotherapy and MCAo-alone group. Combination treatment 24h post-stroke significantly increased the perimeter of von Willebrand factor (vWF)-positive vessels, the diameter and density of alpha smooth muscle actin (αSMA)-positive arteries, and the percentage of 5-bromodeoxyuridine (BrdU)-positive endothelial cells (ECs) in the ischemic boundary zone (IBZ) compared with MCAo-alone or HUCBC-monotherapy 14days after MCAo (p<0.05, n=8/group); Combination treatment significantly increased the densities of vWF-vessels and αSMA-arteries as well as the densities of BrdU-ECs and BrdU-positive smooth muscle cells (SMCs) in vascular walls in the IBZ compared with Simvastatin-monotherapy. Moreover, the increased BrdU-ECs and BrdU-SMCs were significantly correlated with neurological functional outcome 14days after MCAo. Combination treatment also significantly increased the expression of Angiopoietin-1 (Ang1), Tie2 and Occludin in the IBZ (p<0.05, n=8/group). The in vitro experiments showed that combination treatment and Ang1 significantly increased capillary-like tube formation and arterial cell migration; anti-Ang1 significantly reduced combination treatment-induced tube-formation and artery cell migration (p<0.05, n=6/group). These findings indicated that a combination of sub-therapeutic doses of Simvastatin and HUCBCs treatment of stroke increases Ang1/Tie2 and Occludin expression in the ischemic brain, amplifies endogenous angiogenesis and arteriogenesis, and enhances vascular remodeling which in concert may contribute to functional outcome after stroke.Neuroscience 10/2012; 227. DOI:10.1016/j.neuroscience.2012.09.066 · 3.33 Impact Factor