T-cell receptor mimic (TCRm) antibody therapeutics against intracellular proteins.

T-cell receptor mimic (TCRm) antibodies combine the capacity of a T cell to target intracellular antigens with other capacities unique to antibodies. Neoantigens are abnormal proteins that arise as a consequence of somatic mutations. Technological advances promote the development of neoantigen-targeting therapies including TCRm antibody therapies. This review summarizes key characteristics of TCRm antibodies, in particular those targeting neoantigens, and further introduces discussion of obstacles that must be overcome to advance TCRm therapeutics.

Increased Expression of EGR-1 in Diabetic Human Adipose Tissue-Derived Mesenchymal Stem Cells Reduces Their Wound Healing Capacity.

The prevalence of type 2 diabetes mellitus (T2DM), which leads to diabetic complications, has been increasing worldwide. The possible applications of T2DM-derived stem cells in cell therapy are limited because their characteristics are still not fully understood. In this study, we characterized adipose tissue-derived mesenchymal stem cells (AT-MSCs) from diabetic patients (dAT-MSCs) and found that insulin receptor substrate-1 (IRS-1) was highly phosphorylated at serine 636/639 in dAT-MSCs. Moreover, we found that early growth response factor-1 (EGR-1) and its target genes of PTEN and GGPS1 were highly expressed in dAT-MSCs in comparison to healthy donor-derived AT-MSCs (nAT-MSCs). We observed impaired wound healing after the injection of dAT-MSCs in the ischemic flap mouse model. The expressions of EGR-1 and its target genes were diminished by small hairpin RNA-targeted EGR-1 (shEGR-1) and treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibitor (PD98059). Importantly, dAT-MSCs with shEGR-1 were able to restore the wound healing ability in the mouse model. Interestingly, under hypoxic conditions, hypoxia-inducible factor-1α (HIF-1α) can bind to the EGR-1 promoter in dAT-MSCs, but not in nAT-MSCs. Together, these results demonstrate that the expression of EGR-1 was upregulated in dAT-MSCs through two pathways: the main regulatory pathway is the MAPK/ERK pathway, the other is mediated by HIF-1α through direct transcriptional activation at the promoter region of the EGR1 gene. Our study suggests that dAT-MSCs may contribute to microvascular damage and delay wound healing through the overexpression of EGR-1. Interrupting the expression of EGR-1 in dAT-MSCs may be a useful treatment for chronic wounds in diabetic patients.

Dual functions of hypoxia-inducible factor 1 alpha for the commitment of mouse embryonic stem cells toward a neural lineage.

Embryonic stem (ES) cells are useful for elucidating the molecular mechanisms of cell fate decision in the early development of mammals. It has been shown that aggregate culture of ES cells efficiently induces neuroectoderm differentiation. However, the molecular mechanism that leads to selective neural differentiation in aggregate culture is not fully understood. Here, we demonstrate that the oxygen-sensitive hypoxia-inducible transcription factor, Hif-1α, is an essential regulator for neural commitment of ES cells. We found that a hypoxic environment is spontaneously established in differentiating ES cell aggregates within 3 days, and that this time window coincides with Hif-1α activation. In ES cells in adherent culture under hypoxic conditions, Hif-1α activation was correlated with significantly greater expression of neural progenitor-specific gene Sox1 compared with ES cells in adherent culture under normoxic conditions. In contrast, Hif-1α-depleted ES cell aggregates showed severe reduction in Sox1 expression and maintained high expression of undifferentiated ES cell marker genes and epiblast marker gene Fgf5 on day 4. Notably, chromatin immune precipitation assay and luciferase assay showed that Hif-1α might directly activate Sox1 expression. Of additional importance is our finding that attenuation of Hif-1α resulted in an increase of BMP4, a potent inhibitor of neural differentiation, and led to a high level of phosphorylated Smad1. Thus, our results indicate that Hif-1α acts as a positive regulator of neural commitment by promoting the transition of ES cell differentiation from the epiblast into the neuroectoderm state via direct activation of Sox1 expression and suppressing endogenous BMP signaling.

Review of biophysical factors affecting osteogenic differentiation of human adult adipose-derived stem cells.

Developing bone is subject to the control of a broad variety of influences in vivo. For bone repair applications, in vitro osteogenic assays are routinely used to test the responses of bone-forming cells to drugs, hormones, and biomaterials. Results of these assays are used to predict the behavior of bone-forming cells in vivo. Stem cell research has shown promise for enhancing bone repair. In vitro osteogenic assays to test the bone-forming response of stem cells typically use chemical solutions. Stem cell in vitro osteogenic assays often neglect important biophysical cues, such as the forces associated with regular weight-bearing exercise, which promote bone formation. Incorporating more biophysical cues that promote bone formation would improve in vitro osteogenic assays for stem cells. Improved in vitro osteogenic stimulation opens opportunities for "pre-conditioning" cells to differentiate towards the desired lineage. In this review, we explore the role of select biophysical factors-growth surfaces, tensile strain, fluid flow and electromagnetic stimulation-in promoting osteogenic differentiation of stem cells from human adipose. Emphasis is placed on the potential for physical microenvironment manipulation to translate tissue engineering and stem cell research into widespread clinical usage.

Characterization of the laser-based release of micropallets from arrays.

The micropallet array system uses a pulsed laser to release pallets tens of microns to hundreds of micrometers in size from a larger array, enabling selective isolation of single cells adherent to the pallets. We characterize the laser-based release of pallets with respect to pallet array and laser parameters. The threshold laser energy required for pallet release increases linearly with the area of the pallet in contact with the underlying glass substrate. The spacing of the pallets within an array as well as the thickness or height of the pallet does not impact the energy required to release a pallet. Delivery of multiple laser pulses decreases the energy/pulse required for pallet release when the pallets were 100 microm or greater on a side. In addition to the square pallets, complex structures such as cantilevers and spirals could be released without damage using the pulsed laser. Identification of the pallet-array variables influencing the energy required for pallet release as well as strategies to minimize this energy will prove critical in optimizing the release of pallets with cells on the arrays.

Micropallet arrays with poly(ethylene glycol) walls.

Arrays of releasable micropallets with surrounding walls of poly(ethylene glycol) (PEG) were fabricated for the patterning and sorting of adherent cells. PEG walls were fabricated between the SU-8 pallets using a simple, mask-free strategy. By utilizing the difference in UV-transmittance of glass and SU-8, PEG monomer was selectively photopolymerized in the space surrounding the pallets. Since the PEG walls are composed of a cross-linked structure, the stability of the walls is independent of the pallet array geometry and the properties of the overlying solution. Even though surrounded with PEG walls, the individual pallets were detached from the array by the mechanical force generated by a focused laser pulse, with a release threshold of 6 microJ. Since the PEG hydrogels are repellent to protein adsorption and cell attachment, the walls localized cell growth to the pallet top surface. Cells grown in the microwells formed by the PEG walls were released by detaching the underlying pallet. The released cells/pallets were collected, cultured and clonally expanded. The micropallet arrays with PEG walls provide a platform for performing single cell analysis and sorting on chip.

Micropallet arrays for the separation of single, adherent cells.

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.