Abstract
We present methods to characterize mesenchymal stromal cells (MSC) over long time periods in vitro. The methods entail passaging cells multiple times and performing differentiation studies with the cells at each passage. Using an array of surface markers and flow cytometric quantification, the data can be correlated to traditional measures of differentiation such as PCR and staining. Using these methods to quantify the amount of differentiation, we concluded that many common MSC markers do not specifically define MSCs with true stem cell properties. Additionally, adipose-derived as opposed to bone marrow-derived MSCs show long-term CD34+ labeling. The methods described can be used to help identify stem cell markers and to characterize the state of stem cells in vitro. Compiling these data from multiple laboratories would be helpful to determine source, extraction and culture methods needed to obtain high yields of useful stem cells.
Highlights
In the rapidly evolving field of tissue engineering and regenerative medicine, human bone marrow-derived mesenchymal stromal cells have been extensively studied with respect to their differentiation poten-tial towards adipocytes, osteoblasts and chondrocytes, among other tissues, and provide an essential source for these tissue outcomes [1]
An aspirate volume of 25 mL was diluted 10-fold with mesenchymal stromal cells (MSC) expansion medium consisting of DMEM:F12 basal medium (DMEM:F12) supplemented with 10% fetal bovine serum (FBS), antibiotics-antimycotics (100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL fungizone), 0.1 mM nonessential amino acids and 1 ng/mL basic fibroblast growth factor (Invitrogen)
Our method of correlating quantitative flow cytometric data with current standard qualitative assays displays a straightforward and relatively inexpensive approach for detailed characterization of a batch of stem cells. To our knowledge this is the first collection of such detailed analysis of human bone marrow-derived mesenchymal stromal cells (hMSCs) and have shown that adipose-derived stem cells (hASCs) in long-term in vitro culture and will be helpful to aid other researchers in planning future experiments
Summary
In the rapidly evolving field of tissue engineering and regenerative medicine, human bone marrow-derived mesenchymal stromal cells (hMSCs) have been extensively studied with respect to their differentiation poten-tial towards adipocytes, osteoblasts and chondrocytes, among other tissues, and provide an essential source for these tissue outcomes [1]. In the rapidly evolving field of tissue engineering and regenerative medicine, human bone marrow-derived mesenchymal stromal cells (hMSCs) have been extensively studied with respect to their differentiation poten-. Studies have shown that adipose-derived stem cells (hASCs) show similar differentiation properties [4]. When comparing methods in different publications, it becomes apparent that a multitude of extraction protocols and culture conditions exist. Though, most of these studies extract hMSCs by adhesion to tissue culture plastic (TCP) as this method is gentle to the cells, relatively inexpensive and easy to perform [5]. Isolation by adherence generally yields a heterogeneous population and a mixed population might not behave in the same manner as a pure isolated cell population
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