Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: a comparison with articular chondrocytes.

Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose-derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three-dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de- and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL-expanded HACs demonstrated better redifferentiation potential than FCS-expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT-PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL-expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation.

In vitro adipogenesis of adipose-derived stem cells in 3D fibrin matrix of low component concentration.

This study evaluated the suitability of adipose-derived stem cells (ASCs) combined with fibrin matrix of variable composition for adipose tissue-equivalent formation in vitro. Therefore, undifferentiated ASCs were embedded in fibrin clots composed of 2 IU/ml thrombin and fibrinogen of varying concentrations (6.25, 12.5 and 25 mg/ml) and kept under control or adipogenic conditions. Fibrin-cell composites were evaluated by scanning electron microscopy, live/dead staining, lactate-dehydrogenase (LDH) assay, quantitative PCR for the adipogenic markers fatty acid binding protein 4 (FABP4), peroxisome proliferative activated receptor-γ (PPARγ) and leptin, leptin ELISA and oil red O staining. Cells were found homogeneously distributed throughout the clot. Their number increased to day 7 (up to 3.62-fold median) and decreased thereafter until day 28. The proliferation was unaffected by fibrinogen concentration in the control. Adipogenic conditions generally yielded higher cell numbers, which were in addition increasing with increasing fibrinogen concentrations. FABP4, PPARγ and leptin mRNA expression was strongly upregulated by adipogenic medium, which was confirmed by the levels of leptin secretion and lipid vesicles formation demonstrated by oil red O staining. When embedded in 25 mg/ml fibrinogen clots, ASCs showed the highest expression levels of FABP4 (up to 629.0-fold), PPARγ (up to 1.6-fold) and leptin (up to 57.9-fold), corroborated by significantly elevated leptin secretion (median 33.29 ng/ml) on day 14. Constructs composed of fibrin matrix of low component concentrations-allowing homogeneous cell distribution-with ASCs should represent a suitable strategy for adipose tissue formation in vivo.

Isolation of pig bone marrow mesenchymal stem cells suitable for one-step procedures in chondrogenic regeneration.

Large animals such as pigs are good models for skeletal tissue engineering, since they provide physical forces similar to those of humans. Porcine bone marrow mesenchymal stem cells (BMSCs) have shown regenerative capacity similar to those of human BMSCs and can therefore be preclinically applied in settings corresponding to autologous transplantation in patients. Aiming at a one-step procedure for cartilage regeneration with autologous BMSCs, three straightforward isolation methods for BMSCs of Göttingen minipigs were compared. For this purpose, the BMSC fraction was enriched by red blood cell (RBC) lysis, dextran sedimentation or density gradient centrifugation. Isolated BMSCs were evaluated with regard to cell yield, proliferation capacity, phenotype and ability to differentiate to the chondrogenic lineage. Highest cell yields determined at the time of subcultivation were obtained using RBC lysis. In comparison, dextran sedimentation was less efficient but superior to density gradient centrifugation, which yielded significantly lower cell numbers than RBC lysis. The evaluated isolation methods resulted in cultures with equal proliferative capacity, with constant population doubling times of 50-55 h for at least 100 days (approximating to 40 cumulative population doublings) in vitro. Chondrogenic differentiation in micromass pellet cultures was evaluated by glycosaminoglycan quantification, histological staining with Alcian blue and safranin O and immunohistochemical analysis for collagen type II. These evaluations demonstrated that all three isolation methods yielded cells capable of generating cartilaginous tissue in vitro. According to our data, RBC lysis can be used to efficiently isolate porcine BMSCs in a short time frame which would allow for intraoperative one-step procedures in preclinical cartilage regeneration studies.