No abstract

Orthopedic implants including engineered bone tissue are commonly tested in sheep. To avoid rejection of heterologous or xenogeneic cells, autologous cells are preferably used, that is, ovine mesenchymal stem cells (oMSC). Unlike human MSC, ovine MSC are not well studied regarding isolation, expansion, and characterization. Here we investigated the impact of culture media composition on growth characteristics, differentiation, and surface antigen expression of oMSC. The culture media varied in fetal calf serum (FCS) content and in the addition of supplements and/or additional epidermal growth factor (EGF). We found that FCS strongly influenced oMSC proliferation and that specific combinations of supplemental factors (MCDB-201, ITS-plus, dexamethasone, and L-ascorbic acid) determined the expression of surface epitopes. We compared two published protocols for oMSC differentiation towards the osteogenic, adipogenic, and chondrogenic fate and found (i) considerable donor to donor variations, (ii) protocol-dependent variations, and (iii) variations resulting from the preculture medium composition. Our results indicate that the isolation and culture of oMSC in different growth media are highly variable regarding oMSC phenotype and behaviour. Furthermore, variations from donor to donor critically influence growth rate, surface marker expression, and differentiation.

Galectins,β-galactoside binding proteins, function in several physiological and pathological processes. The further evaluation of these processes as well as possible applications of galectins in diagnosis and therapy has raised high scientific interest. Therefore, easy and reliable test systems are necessary. Here we present the simple and cost-efficient production of recombinant human galectins as fusion proteins with SNAP-tag and fluorescent proteins. These constructs show binding specificities and oligomerisation properties generally comparable to recombinant galectins. Their direct fluorescence signal was utilised by ELISA-type assay and flow cytometry analysis with human and ovine mesenchymal stem cells (MSC). Flow cytometry demonstrated glycan mediated binding of His6-SNAP-YFP-Gal- 3 to both MSC types, which was specifically inhibited by lactose. Moreover, directed immobilisation by SNAP-tag technology onto benzylguanine- activated sepharose was utilised to prepare galectin affinity columns for glycoprotein analysis and purification. The SNAPtag directed coupling yielded up to three-fold higher binding capacities for the glycoprotein standard asialofetuin compared to nondirected coupled galectin suggesting improved functionality following directed coupling.

Cord blood-derived haematopoietic stem cells (CB-HSCs) are an attractive source for transplantation in haematopoietic disorders. However, the yield of CB-HSCs per graft is limited and often insufficient, particularly for the treatment of adult patients. Here we compare the capacity of three cytokine cocktails to expand CB-CD34(+) cells. Cells were cultured for 5 or 14 days in media supplemented with: (a) SCF, FL, IL-3 and IL-6 (SFLIL3/6); (b) SCF, TPO, FGF-1 and IL-6 (STFIL6); and (c) SCF, TPO, FGF-1, IGFBP2 and Angptl-5 (STFAI). We observed that STFAI-culture expansion sustained the most vigorous cell proliferation, maintenance of CD34(+) phenotype and colony-forming unit counts. In addition, STFAI-cultured cells had a potent ex vivo migration activity. STFAI-expanded cells were able to engraft NSG mice. However, no significant difference in overall engraftment was observed among the expansion cocktails. Assessment of short-term reconstitution using multilineage markers demonstrated that the STFAI cocktail for HSCs expansion greatly improved total cell expansion but may impair short-term lymphoid repopulation.

Human adult mesenchymal stem cells (hMSC) are particularly suitable cells for autologous tissue engineering and cell-based therapies. They can be isolated from various tissues, such as bone marrow, adipose tissue, dental pulp or umbilical cords. Due to their primitive developmental stage, umbilical cord-derived hMSC are assumed to have a higher proliferation and differentiation capacity than hMSC from adult tissues. We isolated hMSC from bone marrow (BM) and umbilical cords (UC) and compared the cells regarding their surface epitopes, proliferation and differentiation capacity.Flow cytometry of specific surface epitopes showed that both BM-MSC and UC-MSC display the characteristic MSC phenotype. Cells from both sources were readily differentiated into adipocytes, osteoblasts and chondrocytes according to standard protocols. Interestingly, only UC-MSC spontaneously formed three-dimensional aggregates when cultured under post-confluent conditions. The cells of these aggregates were viable and spontaneously differentiated into several specialized cell types akin to the well-known differentiation of embryoid bodies. Besides, UC-MSC expressed the pluripotency-associated gene NANOG as well as genes characteristic for the mesodermal and ectodermal fate. Thus, UC-MSC resemble BM-MSC but additionally show a spontaneous embryoid body-like aggregation and differentiation in vitro. These results indicate that UC-MSC are less restricted than BM-MSC and may thus extend the limits of BM-MSC based therapies. Citation: Adamzyk C, Labude N, Schneider RK, et al. Human umbilical cord-derived mesenchymal stem cells spontaneously form 3d aggregates and differentiate in an embryoid body-like manner. Keywords