Metabolically active human brown adipose tissue derived stem cells.

Brown adipose tissue (BAT) plays a key role in the evolutionarily conserved mechanisms underlying energy homeostasis in mammals. It is characterized by fat vacuoles 5-10 µm in diameter and expression of uncoupling protein one, central to the regulation of thermogenesis. In the human newborn, BAT depots are typically grouped around the vasculature and solid organs. These depots maintain body temperature during cold exposure by warming the blood before its distribution to the periphery. They also ensure an optimal temperature for biochemical reactions within solid organs. BAT had been thought to involute throughout childhood and adolescence. Recent studies, however, have confirmed the presence of active BAT in adult humans with depots residing in cervical, supraclavicular, mediastinal, paravertebral, and suprarenal regions. While human pluripotent stem cells have been differentiated into functional brown adipocytes in vitro and brown adipocyte progenitor cells have been identified in murine skeletal muscle and white adipose tissue, multipotent metabolically active BAT-derived stem cells from a single depot have not been identified in adult humans to date. Here, we demonstrate a clonogenic population of metabolically active BAT stem cells residing in adult humans that can: (a) be expanded in vitro; (b) exhibit multilineage differentiation potential; and (c) functionally differentiate into metabolically active brown adipocytes. Our study defines a new target stem cell population that can be activated to restore energy homeostasis in vivo for the treatment of obesity and related metabolic disorders.

Design and in vitro biocompatibility of a novel ocular drug delivery device.

The capsule drug ring (CDR) is a reservoir and delivery agent, which is designed to be placed within the capsular bag during cataract surgery. Prototypes were manufactured by hot melt extrusion of Bionate II®, a polycarbonate urethane. The devices have been optimized using Avastin® as the drug of interest. In vitro biocompatibility was assessed with human lens epithelial cell (B-3), mouse macrophage (J774A.1) and mouse fibroblast (L-929) cell lines. Cell migration and proliferation were assessed after in vitro culture. Pro-inflammatory cytokines (i.e., MIP-1ß, MIP-1α, MCP-1, IL-1ß, TNF and TGF-ß1) were quantified using cytometric bead array (CBA). Preliminary in vivo biocompatibility and pharmacokinetics testing has been performed in rabbits.

Senescence and quiescence induced compromised function in cultured macrophages.

Implants are predisposed to infection even years after implantation, despite ostensibly being surrounded by innumerable macrophages as part of the host foreign body response. The local implant environment could adversely influence the implant-associated macrophage phenotype, proliferative capacity, activation states, and ability to neutralize pathogens. This study monitored cultured macrophage proliferative states and phagocytotic competence on tissue culture plastic to address the hypothesis that extended contact with foreign materials alters macrophage phenotype. That such macrophage alterations might also occur around implants has significance to the foreign body response, infection, cancer, autoimmune and other diseases. Specifically, multiple indicators of macrophage proliferation in various culture conditions, including cell confluence, long-term culture (21 days), lipopolysaccharide (LPS) stimulation, passaging, and mitogenic stimulation are reported. Importantly, primary murine macrophages became quiescent at high confluence and senescent during long-term culture. Senescent macrophages significantly reduced their ability to phagocytose particles, while quiescent macrophages did not. Cell senescence and quiescence were not observed with repeated passaging. Primary macrophage stimulation with LPS delayed senescence but did not eliminate it. These results prompt the conclusion that both cell quiescence and senescence are observed under common macrophage culture conditions and could alter macrophage behavior and phenotypes in extended in vitro culture, such as the ability to phagocytose. Such macrophage transitions around foreign bodies in vivo are not documented: quiescence and senescence reported here in macrophage culture could be relevant to macrophage behavior both in vitro in bioassays and in vivo in the foreign body response and implant-centered infection.

Demineralized bone matrix as a vehicle for delivering endogenous and exogenous therapeutics in bone repair.

As a unique human bone extract approved for implant use, demineralized bone matrix (DBM) retains substantial amounts of endogenous osteoconductive and osteoinductive proteins. Commercial preparations of DBM represent a clinically accessible, familiar, widely used and degradable bone-filling device, available in composite solid, strip/piece, and semi-solid paste forms. Surgically placed and/or injected, DBM releases its constituent compounds to bone sites with some evidence for inducing new bone formation and accelerating healing. Significantly, DBM also has preclinical history as a drug carrier by direct loading and delivery of several important classes of therapeutics. Exogenous bioactive agents, including small molecule drugs, protein and peptide drugs, nucleic acid drugs and transgenes and therapeutic cells have been formulated within DBM and released to bone sites to enhance DBM's intrinsic biological activity. Local release of these agents from DBM directly to surgical sites in bone provides improved control of dosing and targeting of both endogenous and exogenous bioactivity in the context of bone healing using a clinically familiar product. Given DBM's long clinical track record and commercial accessibility in standard forms and sources, opportunities to formulate DBM as a versatile matrix to deliver therapeutic agents locally to bone sites in orthopedic repair and regenerative medicine contexts are attractive.

Multinucleated giant cells from fibroblast cultures.

Many multinucleated giant cells are well-known to form from macrophage origin. Those formed from other cell types are less described, but may be as prevalent in pathological tissue. Giant multinucleated cells derived from secondary and primary fibroblast sources in various cultures with similar characteristics to foreign body giant cells are reported. Secondary-transformed NIH 3T3 fibroblasts rapidly fuse within 24 h in contact co-cultures with RAW 264.7 immortalized macrophages, while 3T3 monocultures, non-contact (transwell) co-cultures, and macrophage-conditioned media-treated 3T3 monocultures all do not fuse. Primary-derived murine fibroblasts also form multinucleated cells, both in the presence or absence of co-cultured macrophages that increase during long-term culture (5-30 days). In contrast to 3T3 fusion, this primary cell phenomenon is not due to fibroblast fusion, but rather to nuclear division without cytokinesis. That these multinucleated fibroblasts can originate via different mechanisms may influence and distinguish their behaviors in conditions under which they may arise, including various in vitro culture assays, and in certain fibroblastic pathologies such as the foreign body response, fibrosis, cancer and aged tissue.

Cell-cell signaling in co-cultures of macrophages and fibroblasts.

The foreign body response (FBR) comprises a general, ubiquitous host tissue-based reaction to implanted materials. In vitro cell-based models are frequently employed to study FBR mechanisms involving cell signaling responses to materials. However, these models often study only one cell type, identify only limited signals, and cannot accurately represent the complexity of in vivo inflammatory signaling. To address this issue, a cell co-culture system involving two primary effector cells of the FBR, macrophages and fibroblasts, was employed. Cell-cell signaling systems were monitored between these cell types, including long-term 1) culture of one cell type in conditioned media from the other cell type, 2) non-contacting cell co-cultures (paracrine signaling), and 3) contact co-cultures (juxtacrine signaling) of primary- and secondary-derived cells. Cell culture media and cell images were collected on Days 1, 2, 3, 7, 14, and 21 and changes in soluble protein secretion, cellular behavior, and morphology were assessed. Primary- and secondary-derived cells responded uniquely during each signaling scenario and to one another. In general higher in vitro fidelity to FBR-like responses was found in primary cell co-cultures compared to their mono-cultures and all secondary cell cultures.