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Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and "stemness". Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼ 1.6 ×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼ 2.2 ×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multipotent stromal cells (MSCs) can be differentiated into osteoblasts and chondrocytes, making these cells candidates to regenerate cranio-facial injuries and lesions in long bones. A major problem with cell replacement therapy, however, is the loss of transplanted MSCs at the site of graft. Reactive oxygen species (ROS) and nonspecific inflammation generated at the ischemic site have been hypothesized to lead to MSCs loss; studies in vitro show MSCs dying both in the presence of ROS or cytokines like FasL. We questioned whether MSCs themselves may be the source of these death inducers, specifically whether MSCs produce ROS under cytokine challenge. On treating MSCs with FasL, we observed increased ROS production within 2 h, leading to apoptotic death after 6 h of exposure to the cytokine. N-acetyl cysteine, an antioxidant, is able to protect MSCs from FasL-induced ROS production and subsequent ROS-dependent apoptosis, though the MSCs eventually succumb to ROS-independent death signaling. Epidermal growth factor (EGF), a cell survival factor, is able to protect cells from FasL-induced ROS production initially; however, the protective effect wanes with continued FasL exposure. In parallel, FasL induces upregulation of the uncoupling protein UCP2, the main uncoupling protein in MSCs, which is not abrogated by EGF; however, the production of ROS is followed by a delayed apoptotic cell death despite moderation by UCP2. FasL-induced ROS activates the stress-induced MAPK pathways JNK and p38MAPK as well as ERK, along with the activation of Bad, a proapoptotic protein, and suppression of survivin, an antiapoptotic protein; the latter two key modulators of the mitochondrial death pathway. FasL by itself also activates its canonical extrinsic death pathway noted by a time-dependent degradation of c-FLIP and activation of caspase 8. These data suggest that MSCs participate in their own demise due to nonspecific inflammation, holding implications for replacement therapies.

Biomimetic scaffolds have been proposed as a means to facilitate tissue regeneration by multi-potent stromal cells (MSCs). Effective scaffold colonization requires a control of multiple MSC responses including survival, proliferation, differentiation, and migration. As MSC migration is relatively unstudied in this context, we present here a multi-level approach to its understanding and control, integratively tuning cell speed and directional persistence to achieve maximal mean free path (MFP) of migration. This approach employs data-driven computational modeling to ascertain small molecule drug treatments that can enhance MFP on a given materials substratum. Using poly(methyl methacrylate)-graft-poly(ethylene oxide) polymer surfaces tethered with epidermal growth factor (tEGF) and systematically adsorbed with fibronectin, vitronectin, or collagen-I to present hTERT-immortalized human MSCs with growth factor and extracellular matrix cues, we measured cell motility properties along with signaling activities of EGFR, ERK, Akt, and FAK on 19 different substrate conditions. Speed was consistent on collagen/tEGF substrates, but low associated directional persistence limited MFP. Decision tree modeling successfully predicted that ERK inhibition should enhance MFP on collagen/tEGF substrates by increasing persistence. Thus, we demonstrated a two-tiered approach to control MSC migration: materials-based "coarse-graining" complemented by small molecule "fine-tuning"., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs.

MSC can act as a pluripotent source of reparative cells during injury and therefore have great potential in regenerative medicine and tissue engineering. However, the response of MSC to many growth factors and cytokines is unknown. Many envisioned applications of MSC, such as treating large defects in bone, involve in vivo implantation of MSC attached to a scaffold, a process that creates an acute inflammatory environment that may be hostile to MSC survival. Here, we investigated cellular responses of MSC on a biomaterial surface covalently modified with epidermal growth factor (EGF). We found that surface-tethered EGF promotes both cell spreading and survival more strongly than saturating concentrations of soluble EGF. By sustaining mitogen-activated protein kinase kinase-extracellular-regulated kinase signaling, tethered EGF increases the contact of MSC with an otherwise moderately adhesive synthetic polymer and confers resistance to cell death induced by the proinflammatory cytokine, Fas ligand. We concluded that tethered EGF may offer a protective advantage to MSC in vivo during acute inflammatory reactions to tissue engineering scaffolds. The tethered EGF-modified polymers described here could be used together with structural materials to construct MSC scaffolds for the treatment of hard-tissue lesions, such as large bony defects. Disclosure of potential conflicts of interest is found at the end of this article.

Bone graft performance can be enhanced by addition of connective tissue progenitors (CTPs) from fresh bone marrow in a manner that concentrates the CTP cell population within the graft. Here, we used small peptide adhesion ligands presented against an otherwise adhesion-resistant synthetic polymer background in order to illuminate the molecular basis for the attachment and colony formation by osteogenic CTPs from fresh human marrow, and contrast the behavior of fresh marrow to many commonly used osteogenic cell sources. The linear GRGDSPY ligand was as effective as tissue culture polystyrene in fostering attachment of culture-expanded porcine CTPs. Although this GRGDSPY peptide was more effective than control peptides in fostering alkaline phosphatase (AP)-positive colony formation from primary human marrow in 5 of the 7 patients tested, GRGDSPY was as effective as the control glass substrate in only one patient of 7. Thus, the peptide appears capable of enabling osteoblastic development from only a subpopulation of CTPs in marrow. The bone sialoprotein-derived peptide FHRRIKA was ineffective in fostering attachment of primary culture-expanded pig CTPs, although it was as effective as GRGDSPY in fostering AP-positive colonies from fresh human marrow. This study provides insights into integrin-mediated behaviors of CTPs and highlights differences between freshly isolated marrow and culture-expanded cells.

Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-gamma in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs.

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a 'membrane bilayer' microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses. [ABSTRACT FROM AUTHOR]