Expression Of Typical Surface Markers Research Articles

Use of collagenase to isolate adipose tissue-derived stem cells – substantial manipulation or not?

Background: Collagenase is commonly used to isolate the stromal vascular fraction (SVF) or adipose tissue-derived stem cells (ADSCs) from human adipose tissue. Enzymatic breakdowns may be a substantial manipulation according to the classifications of medical regulatory authorities. This study investigates the possible effects of human adipose tissue dissociation with collagenase on in vitro function and behavior of ADSCs. Methods and results: Adipose tissue from nine donors was divided into two equal fractions. SVF was then isolated either mechanically or with collagenase, respectively. The resulting cells were analyzed for their surface markers directly after isolation and at passage five. Proliferation, tri-lineage differentiation, and secretome markers were measured after passage four. Using collagenase compared to mechanical isolation did not alter the expression of typical surface markers of ADSCs. ADSCs isolated with collagenase showed a significantly shorter population doubling time (p < 0.001), a significantly higher mean specific GPDH-activity, a stronger intensity in perilipin staining (p = 0.005), and a significantly higher extracellular calcium deposition (p = 0.006) than mechanically isolated ADSCs. The expression of adipogenic and osteogenic marker genes was not different in mechanically versus enzymatically isolated ADSCs. There were no significant differences in proteoglcyan production (p > 0.05) and the concentration of type 2 collagen. Except for an increased CCL2 concentration in mechanically isolated ASDCs (p = 0.01), there were no significant differences in the concentration of secreted proteins between both isolation methods. Conclusions: The use of collagenase does not substantially impair central in vitro characteristics and functions of human adipose tissue-derived stem cells.

Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem/progenitor cells that can be isolated from various tissues and have attracted increasing attention from the scientific community. This is due to MSCs showing great potential for incurable disease treatment, and most applications of MSCs involve tissue degeneration and treatment of immune- and inflammation-mediated diseases. Conventional MSC cultures contain fetal bovine serum (FBS), which is a common supplement for cell development but is also a risk factor for exposure to animal-derived pathogens. To avoid the risks resulting from the xenogeneic origin and animal-derived pathogens of FBS, xeno-free media have been developed and commercialized to satisfy MSC expansion demands for human clinical applications. This review summarized and provided an overview of xeno-free media that are currently used for MSC expansion. Additionally, we discussed the influences of different xeno-free media on MSC biology with particular regard to cell morphology, surface marker expression, proliferation, differentiation and immunomodulation. The xeno-free media can be serum-free and xeno-free media or media supplemented with some human-originating substances, such as human serum, human platelet lysates, human umbilical cord serum/plasma, or human plasma-derived supplements for cell culture medium. These media have capacity to maintain a spindle-shaped morphology, the expression of typical surface markers, and the capacity of multipotent differentiation and immunomodulation of MSCs. Xeno-free media showed potential for safe use for human clinical treatment. However, the influences of these xeno-free media on MSCs are various and any xeno-free medium should be examined prior to being used for MSC cultures.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease.

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair. Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

Induced differentiation and molecular characterization of monocyte-derived multipotential cells (MOMCs) generated from commonly discarded leukapheresis filters

Background & Aim Μonocyte-derived multipotential cells (MOMCs), easily isolated from the peripheral blood, include progenitors capable of differentiating into endothelial lineages in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis (Kuwana M et al, 2012). Methods, Results & Conclusion discarded leukapheresis filters (T.P.H. Meyer et al, 2005)and estimate the expression of several stem cell factors that play a key role in their pluripotency and differentiation capacity. A leukocyte elution medium composed of Citrate phosphate dextrose (CPD), hypertonic SAGM and Phosphate Buffered Saline was prepared and used for back-flushing of the filters, opposite to the primary blood flow during the leukocyte depletion step. Upon centrifugation in density gradient, mononuclear cells were collected and subsequently incubated in 37οC 5% CO2conditions for 10 days in poly-L-lysine+gelatin coated plates in the presence of 50 ng/ml SDF-1a chemotactic factor enabling the MOMCs generation. Aiming the characterization of the harvested cells, flow cytometry was performed for the detection of typical surface marker expression (CD45, CD14, CD34, Collagen type I and CD184). The induced differentiation capacity of MOMCs towards bone and endothelium in specific culture mediums was estimated via alizarin red staining and flow cytometry, respectively. Finally, the expression levels of genes related with self-renewal and differentiation were estimated with Real-time PCR. The generated MOMCs were characterized by high expression of the CD45/CD14/CD34/CD184/Col I markers while progressively lost their native markers and gained endothelial or bone features upon induced differentiation. The MOMCs expressed several stem cell factors like Nanog, Oct4, c-Myc, Sox2, Klf4, Sall4, ESRRB and ZNF214. All these factors are responsible to activate the networks related with the pluripotency, differentiation and reprogramming of cells while are highly expressed in embryonic stem cells. To conclude, we successfully optimized conditions to elute leukocytes adsorbed to leukapheresis filters resulting in leukocyte suspensions collection for multiple uses. The easy and quick generation of MOMCs from monocytes derived from leukapheresis filters, makes them potential candidates for future therapeutic application in tissue regeneration.

A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid

Background/PurposeExosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. MethodsExosomes were isolated by reagent-enhanced centrifugation from cell culture media of banked human bone marrow (bm) and amniotic fluid (af) MSCs after serum starvation. Characterization was by flow exometry for tetraspanin markers CD9, CD63, and CD81, transmission electron microscopy for size and morphology, and tunable resistive pulse sensing for size distribution and concentration. Statistical comparisons of count data were made by Poisson regression modeling and Student's T-test. ResultsExosomes of appropriate size and morphology were isolated with comparable expressions of CD9 (96% vs. 94%), CD63 (88% vs. 66%), and CD81 (71% vs. 63%) for bmMSC and afMSC, respectively. Total exosome yield (particles/mL) adjusted for number of cells was higher from afMSCs than bmMSCs by an estimated 25% (P < 0.001). ConclusionsWhile bone marrow and amniotic fluid mesenchymal stem cells are comparable sources of exosomes in size distribution, morphology, and expression of typical surface markers, yield may be higher from amniotic fluid cells. The amniotic fluid appears to be a preferable source of exosomes for clinical applications. Level of evidenceN/A (bench laboratory study).