Chronic skin wounds are characterized by poor re-epithelialization, angiogenesis and granulation. Previous work has demonstrated that topical stromal cell-derived growth factor-1 (SDF1) promotes ...neovascularization, resulting in faster re-epithelialization of skin wounds in diabetic mice. However, the clinical usefulness of such bioactive peptides is limited because they are rapidly degraded in the wound environment due to high levels of proteases. Here, we describe the development of a recombinant fusion protein comprised of SDF1 and an elastin-like peptide that confers the ability to self-assemble into nanoparticles. The fusion protein and recombinant human SDF1 showed similar binding characteristics, as indicated by the measured equilibrium dissociation constant (Kd) for the binding of free SDF1 or the fusion protein to the CXCR4 receptor. The biological activity of SDF1-ELP, as measured by intracellular calcium release in HL60 cells was dose dependent, and also very similar to that of free SDF1. In contrast, the biological activity of SDF1-ELP in vivo was significantly superior to that of free SDF1. When applied to full thickness skin wounds in diabetic mice, wounds treated with SDF1-ELP nanoparticles were 95% closed by day 21, and fully closed by day 28, while wounds treated with free SDF1, ELP alone, or vehicle were only 80% closed by day 21, and took 42days to fully close. In addition, the SDF1-ELP nanoparticles significantly increased the epidermal and dermal layer of the healed wound, as compared to the other groups. These results indicate that SDF1-ELP fusion protein nanoparticles are promising agents for the treatment of chronic skin wounds.
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MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be ...undiscovered microRNAs expressed early in stem cell differentiation.
SOLiD ultra-deep sequencing identified >10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.
Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.
BackgroundIn a prior report, we detailed the isolation and engineering of a bispecific killer cell engager, referred to as BiKE:E5C1. The BiKE:E5C1 exhibits high affinity/specificity for the CD16a ...activating receptor on natural killer (NK) cells and human epidermal growth factor receptor 2 (HER2) on cancer cells. In vitro studies have demonstrated that BiKE:E5C1 can activate the NK cells and induce the killing of HER2+ ovarian and breast cancer cells, surpassing the performance of the best-in-class monoclonal antibody, Trazimera (trastuzumab). To advance this BiKE technology toward clinical application, the objective of this research was to demonstrate the ability of BiKE:E5C1 to activate CD16+ immune cells such as NK cells and macrophages to kill cancer cells, and eradicate metastatic HER2+ tumors in NK humanized NOG mice.MethodsWe assessed BiKE:E5C1’s potential to activate CD16-expressing peripheral blood (PB)-NK cells, laNK92 cells, and THP-1-CD16A monocyte-macrophages through flowcytometry and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC) assays. Subsequently, laNK92 cells were selected as effector cells and genetically modified to express the nanoluciferase gene, enabling the monitoring of their viability in NK humanized NOG mice using quantitative bioluminescent imaging (qBLI). To evaluate the functionality of BiKE:E5C1 in vivo, we introduced firefly luciferase-expressing ovarian cancer cells via intraperitoneal injection into hIL-15 and hIL-2 NOG mice, creating a model of ovarian cancer metastasis. Once tumor establishment was confirmed, we treated the mice with laNK92 cells plus BiKE:E5C1 and the response to therapy was assessed using qBLI.ResultsOur data demonstrate that BiKE:E5C1 activates not only laNK92 cells but also PB-NK cells and macrophages, significantly enhancing their anticancer activities. ADCC assay demonstrated that IgG1 Fc region had no impact on BiKE:E5C1’s anticancer activity. In vivo results reveal that both hIL-15 and hIL-2 NOG mouse models support the viability and proliferation of laNK92 cells. Furthermore, it was observed that BiKE:E5C1 activates laNK92 cells in mice, leading to eradication of cancer metastasis in both NK humanized hIL-15 and hIL-2 NOG mouse models.ConclusionsCollectively, our in vivo findings underscore BiKE:E5C1’s potential as an immune cell engager capable of activating immune cells for cancer cell elimination, thereby expanding the arsenal of available BiKEs for cancer immunotherapy.
Cell replacement therapy is a promising treatment strategy for Parkinson's disease (PD); however, the poor survival rate of transplanted neurons is a critical barrier to functional recovery. In this ...study, we used self-assembling peptide nanofiber scaffolds (SAPNS) based on the peptide RADA16-I to support the
maturation and
post-transplantation survival of encapsulated human dopaminergic (DA) neurons derived from induced pluripotent stem cells. Neurons encapsulated within the SAPNS expressed mature neuronal and midbrain DA markers and demonstrated
functional activity similar to neurons cultured in two dimensions. A microfluidic droplet generation method was used to encapsulate cells within monodisperse SAPNS microspheres, which were subsequently used to transplant adherent, functional networks of DA neurons into the striatum of a 6-hydroxydopamine-lesioned PD mouse model. SAPNS microspheres significantly increased the
survival of encapsulated neurons compared with neurons transplanted in suspension, and they enabled significant recovery in motor function compared with control lesioned mice using approximately an order of magnitude fewer neurons than have been previously needed to demonstrate behavioral recovery. These results indicate that such biomaterial scaffolds can be used as neuronal transplantation vehicles to successfully improve the outcome of cell replacement therapies for PD. Impact Statement Transplantation of dopaminergic (DA) neurons holds potential as a treatment for Parkinson's disease (PD), but low survival rates of transplanted neurons is a barrier to successfully improving motor function. In this study, we used hydrogel scaffolds to transplant DA neurons into PD model mice. The hydrogel scaffolds enhanced survival of the transplanted neurons compared with neurons that were transplanted in a conventional manner, and they also improved recovery of motor function by using significantly fewer neurons than have typically been transplanted to see functional benefits. This cell transplantation technology has the capability to improve the outcome of neuron transplantation therapies.
ABSTRACT
Substrates used to culture human embryonic stem cells (hESCs) are typically 2‐dimensional (2‐D) in nature, with limited ability to recapitulate in vivo‐like 3‐dimensional (3‐D) ...microenvironments. We examined critical determinants of hESC self‐renewal in poly‐d‐lysine‐pretreated synthetic polymer‐based substrates with variable microgeometries, including planar 2‐D films, macroporous 3‐D sponges, and microfibrous 3‐D fiber mats. Completely synthetic 2‐D substrates and 3‐D macroporous scaffolds failed to retain hESCs or support self‐renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self‐renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell‐cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3‐D microgeometries can support hESC colony formation, self‐renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3‐D and for stem cell differentiation in situ and transplantation using defined humanized conditions.—Carlson, A. L., Florek, C. A., Kim, J. J., Neubauer, T., Moore, J. C., Cohen, R. I., Kohn, J., Grumet, M., Moghe, P. V. Microfibrous substrate geometry as a critical trigger for organization, self‐renewal, and differentiation of human embryonic stem cells within synthetic 3‐dimensional microenvironments. FASEB J. 26, 3240–3251 (2012). www.fasebj.org
After muscle loss or injury, skeletal muscle tissue has the ability to regenerate and return its function. However, large volume defects in skeletal muscle tissue pose a challenge to regenerate due ...to the absence of regenerative elements such as biophysical and biochemical cues, making the development of new treatments necessary. One potential solution is to utilize electroactive polymers that can change size or shape in response to an external electric field. Poly(ethylene glycol) diacrylate (PEGDA) is one such polymer, which holds great potential as a scaffold for muscle tissue regeneration due to its mechanical properties. In addition, the versatile chemistry of this polymer allows for the conjugation of new functional groups to enhance its electroactive properties and biocompatibility. Herein, we have developed an electroactive copolymer of PEGDA and acrylic acid (AA) in combination with collagen methacrylate (CMA) to promote cell adhesion and proliferation. The electroactive properties of the CMA + PEGDA:AA constructs were investigated through actuation studies. Furthermore, the biological properties of the hydrogel were investigated in a 14-day
in vitro
study to evaluate myosin light chain (MLC) expression and metabolic activity of C2C12 mouse myoblast cells. The addition of CMA improved some aspects of material bioactivity, such as MLC expression in C2C12 mouse myoblast cells. However, the incorporation of CMA in the PEGDA:AA hydrogels reduced the sample movement when placed under an electric field, possibly due to steric hindrance from the CMA. Further research is needed to optimize the use of CMA in combination with PEGDA:AA as a potential scaffold for skeletal muscle tissue engineering.
Cell replacement therapy is one potential avenue for central nervous system (CNS) repair. However, transplanted stem cells may not contribute to long-term recovery of the damaged CNS unless they are ...engineered for functional advantage. To fine tune regenerative capabilities, we developed a human neural cell line expressing L1, a regeneration-conducive adhesion molecule, under the control of a doxycycline regulatable Tet-off promoter. Controlled expression of L1 is desired because overexpression after regenerative events may lead to adverse consequences. The regulated system was tested in several cell lines, where doxycycline completely eliminated green fluorescent protein or L1 expression by 3-5 days in vitro. Increased colony formation as well as decreased proliferation were observed in H9NSCs without doxycycline (hL1-on). To test the role of L1 in vivo after acute compression spinal cord injury of immunosuppressed mice, quantum dot labeled hL1-on or hL1-off cells were injected at three sites: lesion; proximal; and caudal. Mice transplanted with hL1-on cells showed a better Basso Mouse Scale score, when compared to those with hL1-off cells. As compared to the hL1-off versus hL1-on cell transplanted mice 6 weeks post-transplantation, expression levels of L1, migration of transplanted cells, and immunoreactivity for tyrosine hydroxylase were higher, whereas expression of chondroitin sulfate proteoglycans was lower. Results indicate that L1 expression is regulatable in human stem cells by doxycycline in a nonviral engineering approach. Regulatable expression in a prospective nonleaky Tet-off system could hold promise for therapy, based on the multifunctional roles of L1, including neuronal migration and survival, neuritogenesis, myelination, and synaptic plasticity.
Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (“mushroom alcohol”), a major fungal ...volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0–1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated “live and dead” assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(−)-1-octen-3-ol. The inhibition concentration 50 (IC
50
) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(−)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC
50
values were 40–80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.
FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in ...development, we have mutated FGFR2 by deleting the entire immunoglobin-like domain III of the receptor. We showed that murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2(DeltaIgIII/DeltaIgIII) embryos displayed two distinct defects that resulted in failures in formation of a functional placenta. About one third of the mutants failed to form the chorioallantoic fusion junction and the remaining mutants did not have the labyrinthine portion of the placenta. Consequently, all mutants died at 10â11 days of gestation. Interestingly, Fgfr2(DeltaIgIII/DeltaIgIII) embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction.
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