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    ABSTRACT: The proliferation and differentiation characteristics of umbilical cord blood mononuclear cells were examined in a non-rotational suspension bioreactor with a fishtail mixer. The system consisted of a glass vessel, a mixer that moved vertically, a data acquisition and control system to continuously monitor pH, temperature and dissolved O(2). The bioreactor provided superior expansion of total HSCs and not total cell number, as well as expression of stemness-related genes which followed with increasing in number of colony-forming cells during 14 days of culture compared to T -lask culture. Vertical agitation thus reduces the total cell number, which may be related to increased shear stress, but has no effect on HSC function.
    Biotechnology Letters 07/2012; 34(11):2125-31.
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    Archives of Iranian medicine 05/2011; 14(3):225-36.
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    ABSTRACT: Despite the suitability of a mouse model for preclinical investigations; little is known regarding mesenchymal stem cells derived from murine amniotic fluid. This is the subject of the present study. Amniotic fluid was collected from NMRI mice during the second weeks of pregnancy and plated. The cells that adhered to the culture surfaces were propagated with three successive subcultures and then characterized. To determine the differentiation potential, the cells were cultivated under osteogenic, adipogenic, and chondrogenic conditions, and followed by specific staining and RT-PCR analysis for differentiation. The proliferative potential of the cells were measured with clonogenic assays, population doubling time and number and by growth curve plotting. Cellular aging was investigated with the senescence-associated ß-galactosidase staining method. The amniotic fluid primary cell culture was composed of round flattened and fibroblastic cells. The latter dominated the culture after several passages. Successful tripotent differentiation of the isolated cells into bone, cartilage and adipose cells were indicative of their mesenchymal stem cells nature. The isolated cells appeared to be relatively proliferative cells as confirmed by the population doubling time value which was equal to about 69 hours. Furthermore, the cells were relatively clonogenic and they tended to initiate proliferation immediately after plating (there was no lag phase in their growth curve). ß-galactosidase positive cells were first observed at passage 3 and increased in number with subsequent passages. Collectively it was concluded that murine amniotic fluid contained mesenchymal stem cells with relatively high proliferation property and typical tripotent differentiation potential.
    Archives of Iranian medicine 03/2011; 14(2):96-103.
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    ABSTRACT: This study was designed to investigate the morphological features of osteogenic cultures that were established from canine marrow derived-mesenchymal stem cells (MSCs). Tripotent canine MSCs were plated in osteogenic conditions for 3 weeks, at the end of which the cultures were observed by light and transmission electron microscopy. Alkaline phosphatase (ALP) activity of the culture was determined during the differentiation period. To assess whether endochondral or intramembranous ossification was involved in MSC bone differentiation, the cultures were explored for cartilage-related gene expression. Multiple nodule-like cell aggregates appeared to form in the osteogenic cultures. These nodules were covered by a periosteum-like layer and osteocyte-like cells of varying morphology were located in lacuna-like cavities within the nodule mass. Furthermore, the bone nodules possessed an abundant matrix in which clearly striated collagen I fibres were arranged in perpendicular bundles. Matrix vesicles involving in matrix mineralization were evident in the nodules. This was in accordance with increased ALP activity in the culture. No expression of cartilage-related genes was observed, which suggested that osteogenesis might occur by intramembranous ossification. In conclusion, canine MSCs could be an appropriate model for studying in vitro bone development.
    Anantomia Histologia Embryologia 06/2010; 39(5):446-55.
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    ABSTRACT: Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing-thawing process usually takes about 2 h excluding overnight incubation at -80 degrees C.
    Nature Protocol 03/2010; 5(3):588-94.
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    ABSTRACT: The aim of this study is to create an ex vivo model to examine the expression of major heat-shock protein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate 70 (HCS70) at the mRNA and protein level in differentiating corneal cells from limbal stem cells (LSC) following air exposure. Limbal biopsies taken from cadaveric normal human limbus were cultivated as explants on human amniotic membrane (HAM) and plastic dish (PD). Corneal differentiation was induced by air lifting for 16 days. The expression of putative LSC markers (P63 and ATP-binding cassette G2 [ABCG2]), corneal markers (keratin 3 [K3/12] and connexin 43 [CX43]), and HSP60, HSP72, HSP90, and HSC70 were tested by RT-PCR, immunofluorescence, and flow cytometry pre- and post-air exposure. Fresh limbal and corneal tissues were used as control groups. Air lifting induced corneal differentiation with a decrease in the number of P63(+) cells and an increase in the number of K3(+)/CX43(+) cells, which characterized transient amplifying cells (TACs). Moreover, denuded HAM provided a superior niche for LSC proliferation and phenotype maintenance in vitro. Additionally, we have evidence that expressions of HSC70 as well as HSP72 were enhanced through corneal differentiation and HSP90 post-air lifting in vitro and in vivo. HSP60, however, was not detected in either LSC or corneal cells, in vivo and in vitro. These results suggest that corneal differentiation following air exposure may regulate HSP72 and HSC70 expression. In addition, HSP72 and HSP90 may protect LSC and corneal cells against oxidative stress.
    Molecular vision 01/2010; 16:1680-8.
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