Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing

Abstract

Human mesenchymal stem cells (hMSC) are a heterogeneous cell population, which is reflected in varying morphological and biological properties. Three subpopulations with intrinsic characteristics can be distinguished: small rapidly self-renewing cells, spindle-shaped cells and large, flattened cells. Unfortunately, it has neither been possible to morphologically define these distinct cells consistently, nor to relate them to specific surface marker features. Here, the primary hMSC subpopulations of three donors are clearly defined by maximum cell diameter and area. Furthermore, these cells were stained for the putative hMSC surface markers CD105, CD90 as well as CD73, and evaluated by three-colour flow cytometry and simultaneous multicolour immunocytochemistry. Interestingly, cell cultures with a high rate of triple-positive hMSC featured a higher content of rapidly self-renewing cells. On the other hand, a higher fraction of flattened cells correlated with a loss of one or more hMSC surface markers. The expression of CD73 showed the highest heterogeneity. Immunocytochemistry further confirmed that flattened cells mainly lack CD73 expression, whereas rapidly self-renewing cells were steadily positive for all three hMSC markers. In the literature, hMSC properties are especially conceded to rapidly self-renewing cells, whereas flattened cells have been suggested to represent early stages of lineage-specific progenitors. We reveal that among the recently suggested surface markers, CD73 is the most sensitive, as it seems to be down-regulated in the early stages of differentiation. Our morphological and immunocytochemical characterization of hMSC subpopulations indicates the yield of early multipotent hMSC and thereby provides a quality control approach for hMSC culturing.

Cell-Surface Expression of Neuron-Glial Antigen 2 (NG2) and Melanoma Cell Adhesion Molecule (CD146) in Heterogeneous Cultures of Marrow-Derived Mesenchymal Stem Cells

“…There are inconsistencies in the literature regarding the ability of the established MSC markers CD73, CD90, and CD105 12 to detect cell populations in heterogeneous MSC cultures. Higher surface expression in rapidly growing MSCs has been reported for these markers in some cases, 33,34 but not in others. 35,36 In our laboratory, OACs and parental MSCs have similar fluorescence intensity histograms for the three biomarkers, suggesting that other means to isolate MSC populations are warranted.…”

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

“…pre-adipocytes) and 4) cuboidal endothelial cells that are lost with passage 34 . Subsequent studies have shown that the round, rapidly renewing cell population possesses the highest multipotentiality and express typical stem cell markers 35,36 . Moreover, they may form "spheroids" due to their rapid proliferation and are often surrounded by spindle-shaped fibroblasts.…”

Analysis of Biological Properties of Human Adult Mesenchymal Stem Cells and Their Effect on Mouse Hind Limb Ischemia

“…As described in the literature [7, 26, 27], three distinct morphological subpopulations were found in cultured cells from each donor (Fig. 2a): very small rapidly self-renewing cells (RS cells), spindle-shaped cells (SS cells), and large and flattened cells with a prominent nucleus (FC cells).…”

“…The mean cell size from each sample and standard deviation were calculated. In view of the graphical results and based on studies from others authors [7], cells higher than 9,500 µm 3 were considered large cells, while cells under 2,500 µm 3 were considered small cells. The percentages of large, medium, and small cells of the total population were determined.…”