Sourcing of an alternative pericyte-like cell type from peripheral blood in clinically relevant numbers for therapeutic angiogenic applications.

Autologous cells hold great potential for personalized cell therapy, reducing immunological and risk of infections. However, low cell counts at harvest with subsequently long expansion times with associated cell function loss currently impede the advancement of autologous cell therapy approaches. Here, we aimed to source clinically relevant numbers of proangiogenic cells from an easy accessible cell source, namely peripheral blood. Using macromolecular crowding (MMC) as a biotechnological platform, we derived a novel cell type from peripheral blood that is generated within 5 days in large numbers (10-40 million cells per 100 ml of blood). This blood-derived angiogenic cell (BDAC) type is of monocytic origin, but exhibits pericyte markers PDGFR-ß and NG2 and demonstrates strong angiogenic activity, hitherto ascribed only to MSC-like pericytes. Our findings suggest that BDACs represent an alternative pericyte-like cell population of hematopoietic origin that is involved in promoting early stages of microvasculature formation. As a proof of principle of BDAC efficacy in an ischemic disease model, BDAC injection rescued affected tissues in a murine hind limb ischemia model by accelerating and enhancing revascularization. Derived from a renewable tissue that is easy to collect, BDACs overcome current short-comings of autologous cell therapy, in particular for tissue repair strategies.

Simultaneous Delivery of Highly Diverse Bioactive Compounds from Blend Electrospun Fibers for Skin Wound Healing.

Blend emulsion electrospinning is widely perceived to destroy the bioactivity of proteins, and a blend emulsion of water-soluble and nonsoluble molecules is believed to be thermodynamically unstable to electrospin smoothly. Here we demonstrate a method to retain the bioactivity of disparate fragile biomolecules when electrospun. Using bovine serum albumin as a carrier protein; water-soluble vitamin C, fat soluble vitamin D3, steroid hormone hydrocortisone, peptide hormone insulin, thyroid hormone triiodothyronine (T3), and peptide epidermal growth factor (EGF) were simultaneously blend-spun into PLGA-collagen nanofibers. Upon release, vitamin C maintained the ability to facilitate Type I collagen secretion by fibroblasts, EGF stimulated skin fibroblast proliferation, and insulin potentiated adipogenic differentiation. Transgenic cell reporter assays confirmed the bioactivity of vitamin D3, T3, and hydrocortisone. These factors concertedly increased keratinocyte and fibroblast proliferation while maintaining keratinocyte basal state. This method presents an elegant solution to simultaneously deliver disparate bioactive biomolecules for wound healing applications.

Microcapsules engineered to support mesenchymal stem cell (MSC) survival and proliferation enable long-term retention of MSCs in infarcted myocardium.

The limited efficacy of cardiac cell-based therapy is thought to be due to poor cell retention within the myocardium. Hence, there is an urgent need for biomaterials that aid in long-term cell retention. This study describes the development of injectable microcapsules for the delivery of mesenchymal stem cells (MSCs) into the infarcted cardiac wall. These microcapsules comprise of low concentrations of agarose supplemented with extracellular matrix (ECM) proteins collagen and fibrin. Dextran sulfate, a negatively charged polycarbohydrate, was added to mimic glycosaminoglycans in the ECM. Cell viability assays showed that a combination of all components is necessary to support long-term survival and proliferation of MSCs within microcapsules. Following intramyocardial transplantation, microcapsules degraded slowly in vivo and did not induce a fibrotic foreign body response. Pre-labeling of encapsulated MSCs with iron oxide nanoparticles allowed continued cell-tracking by MRI over several weeks following transplantation into infarcted myocardium. In contrast, MSCs injected as cell suspension were only detectable for two days post transplantation by MRI. Histological analysis confirmed integration of transplanted cells at the infarct site. Therefore, microcapsules proved to be suitable for stem cell delivery into the infarcted myocardium and can overcome current limitations of poor cell retention in cardiac cell-based therapy.

Not all MSCs can act as pericytes: functional in vitro assays to distinguish pericytes from other mesenchymal stem cells in angiogenesis.

Pericytes play a crucial role in angiogenesis and vascular maintenance. They can be readily identified in vivo and isolated as CD146(+)CD34(-) cells from various tissues. Whether these and other markers reliably identify pericytes in vitro is unclear. CD146(+)CD34(-) selected cells exhibit multilineage potential. Thus, their perivascular location might represent a stem cell niche. This has spurred assumptions that not only all pericytes are mesenchymal stromal cells (MSCs), but also that all MSCs can act as pericytes. Considering this hypothesis, we developed functional assays by confronting test cells with endothelial cultures based on matrigel assay, spheroid sprouting, and cord formation. We calibrated these assays first with commercial cell lines [CD146(+)CD34(-) placenta-derived pericytes (Pl-Prc), bone marrow (bm)MSCs and fibroblasts]. We then functionally compared the angiogenic abilities of CD146(+)CD34(-)selected bmMSCs with CD146(-) selected bmMSCs from fresh human bm aspirates. We show here that only CD146(+)CD34(-) selected Pl-Prc and CD146(+)CD34(-) selected bmMSCs maintain endothelial tubular networks on matrigel and improve endothelial sprout morphology. CD146(-) selected bmMSCs neither showed these abilities, nor did they attain pericyte function despite progressive CD146 expression once passaged. Thus, cell culture conditions appear to influence expression of this and other reported pericyte markers significantly without correlation to function. The newly developed assays, therefore, promise to close a gap in the in vitro identification of pericytes via function. Indeed, our functional data suggest that pericytes represent a subpopulation of MSCs in bm with a specialized role in vascular biology. However, these functions are not inherent to all MSCs.

Controlled drug release from biodegradable thermoresponsive physical hydrogel nanofibers.

Hydrogel nanofiber mats based on thermoresponsive multiblock poly(ester urethane)s comprising poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), and poly(epsilon-caprolactone) (PCL) segments were fabricated by electrospinning. The hydrogel nanofiber mats were more water absorbent under cold conditions and shrunk when exposed to higher temperatures. The rate of protein release could be controlled by changing the temperature of the nanofiber environment. Cell culture studies on the nanofiber mats were carried out using human dermal fibroblasts, and healthy cell morphology was observed. The adherent viable cells were quantified by MTS after rinsing in excess buffer solution. The results showed that these nanofiber scaffolds supported excellent cell adhesion, comparable with the pure PCL nanofibers. The increased hydrophilicity of these hydrogel nanofiber mats led to a more rapid hydrolytic degradation, compared with the pure PCL nanofiber mats. These hydrogel nanofiber scaffolds could potentially be used as thermoresponsive biodegradable supporting structures for skin tissue engineering applications.