Colocalization of cell death with antigen deposition in skin enhances vaccine immunogenicity.

Vaccines delivered to the skin by microneedles-with and without adjuvants-have increased immunogenicity with lower doses than standard vaccine delivery techniques such as intramuscular or intradermal injection. However, the mechanisms underlying this skin-mediated "adjuvant" effect are not clear. Here, we show that the dynamic application of a microprojection array (the Nanopatch) to skin generates localized transient stresses invoking cell death around each projection. Nanopatch application caused significantly higher levels (∼65-fold) of cell death in murine ear skin than i.d. injection using a hypodermic needle. Measured skin cell death is associated with modeled stresses ∼1-10 MPa. Nanopatch-immunized groups also yielded consistently higher anti-immunoglobulin G endpoint titers (up to 50-fold higher) than i.d. groups after delivery of a split virion influenza vaccine. Importantly, colocalization of cell death with nearby live skin cells and delivered antigen was necessary for immunogenicity enhancement. These results suggest a correlation between cell death caused by the Nanopatch with increased immunogenicity. We propose that the localized cell death serves as a "physical immune enhancer" for the adjacent viable skin cells, which also receive antigen from the projections. This natural immune enhancer effect has the potential to mitigate or replace chemical-based adjuvants in vaccines.

Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies.

High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.

The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells.

Human embryonic stem (hES) cells have enormous potential for clinical applications. However, one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis, which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased, F-actin content and distribution is altered, and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However, exactly how the extrinsic pathway is activated is still unclear and deserves further investigation.

Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation.

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post-thaw culture, the presence of 10 microM ROCK inhibitor (Y-27632) and 1 microM pifithrin-mu together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells.

Combinatorial signaling microenvironments for studying stem cell fate.

Extracellular matrix (ECM) and growth factor signaling networks are known to interact in a complex manner. Therefore, reductionist approaches that test the cellular response to individual ECM components and growth factors cannot be used to predict the response to more complex mixtures without knowledge of the underlying signaling network. To address this challenge, we have developed a technology platform to experimentally probe the interactions of ECM components and soluble growth factors on stem cell fate. We present a multiwell microarray platform that allows 1200 simultaneous experiments on 240 unique signaling environments. Mixtures of extracellular matrix (fibronectin, laminin, collagen I, collagen III, collagen IV) are arrayed using a robotic spotter and arranged in a multiwell format. Embryonic stem (ES) cells adhere to ECM spots and are cultured in mixtures of soluble factors [wnt3a, activin A, bone morphogenetic protein-4 (BMP-4), and fibroblast growth factor-4 (FGF-4)]. Differentiation along the cardiac lineage is monitored by myosin heavy chain-alpha-green fluorescent protein (MHC alpha-GFP) reporter expression as compared to growth by monitoring nuclear DNA, and both signals are quantified using a confocal microarray scanner. In developing the platform, we characterized the amount of deposited protein, the fluorescent readout of GFP expression and DNA content, and the use of a laser-based scanner as compared to fluorescent microscopy for data acquisition. The effects of growth factors on growth and differentiation are consistent with previously reported literature, and preliminary evidence of interactive signaling is illuminated. This versatile technique is compatible with virtually any set of insoluble and soluble cues, leverages existing software and hardware, and represents a step toward developing the 'systems biology' of stem cells.

An extracellular matrix microarray for probing cellular differentiation.

We present an extracellular matrix (ECM) microarray platform for the culture of patterned cells atop combinatorial matrix mixtures. This platform enables the study of differentiation in response to a multitude of microenvironments in parallel. The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. To demonstrate its utility, we applied this platform to study the effects of 32 different combinations of five extracellular matrix molecules (collagen I, collagen III, collagen IV, laminin and fibronectin) on cellular differentiation in two contexts: maintenance of primary rat hepatocyte phenotype indicated by intracellular albumin staining and differentiation of mouse embryonic stem (ES) cells toward an early hepatic fate, indicated by expression of a beta-galactosidase reporter fused to the fetal liver-specific gene, Ankrd17 (also known as gtar). Using this technique, we identified combinations of ECM that synergistically impacted both hepatocyte function and ES cell differentiation. This versatile technique can be easily adapted to other applications, as it is amenable to studying almost any insoluble microenvironmental cue in a combinatorial fashion and is compatible with several cell types.