The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm³ tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing neural and liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.

Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.

Aggregation by design: Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science , this issue p. 10.1126/science.aah4949

The preclinical development of peptidyl drugs for cancer treatment is hampered by their poor pharmacologic properties and cell penetrative capabilities in vivo. In this study, we report a nanoparticle-based formulation that overcomes these limitations, illustrating their utility in studies of the anticancer peptide NuBCP-9, which converts BCL-2 from a cell protector to a cell killer. NuBCP-9 was encapsulated in polymeric nanoparticles composed of a polyethylene glycol (PEG)–modified polylactic acid (PLA) diblock copolymer (NuBCP-9/PLA-PEG) or PEG-polypropylene glycol-PEG-modified PLA—tetrablock copolymer (NuBCP-9/PLA-PEG-PPG-PEG). We found that peptide encapsulation was enhanced by increasing the PEG chain length in the block copolymers. NuBCP-9 release from the nanoparticles was controlled by both PEG chain length and the PLA molecular weight, permitting time-release over sustained periods. Treatment of human cancer cells with these nanoparticles in vitro triggered apoptosis by NuBCP-9–mediated mechanism, with a potency similar to NuBCP-9 linked to a cell-penetrating poly-Arg peptide. Strikingly, in vivo administration of NuBCP-9/nanoparticles triggered complete regressions in the Ehrlich syngeneic mouse model of solid tumor. Our results illustrate an effective method for sustained delivery of anticancer peptides, highlighting the superior qualities of the novel PLA-PEG-PPG-PEG tetrablock copolymer formulation as a tool to target intracellular proteins. Cancer Res; 74(12); 3271–81. ©2014 AACR.

Purpose: The MUC1-C oncoprotein is an intracellular target that is druggable with cell-penetrating peptide inhibitors. However, development of peptidyl drugs for treating cancer has been a challenge because of unfavorable pharmacokinetic parameters and limited cell-penetrating capabilities. Experimental Design: Encapsulation of the MUC1-C inhibitor GO-203 in novel polymeric nanoparticles was studied for effects on intracellular targeting of MUC1-C signaling and function. Results: Our results show that loading GO-203 into tetrablock polylactic acid (PLA)-polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG copolymers is achievable and, notably, is enhanced by increasing PEG chain length. In addition, we found that release of GO-203 from these nanoparticles is controllable over at least 7 days. GO-203/nanoparticle treatment of MUC1-C–positive breast and lung cancer cells in vitro was more active with less frequent dosing than that achieved with nonencapsulated GO-203. Moreover, treatment with GO-203/nanoparticles blocked MUC1-C homodimerization, consistent with on-target effects. GO-203/nanoparticle treatment was also effective in downregulating TIGAR, disrupting redox balance, and inhibiting the self-renewal capacity of cancer cells. Significantly, weekly administration of GO-203/nanoparticles to mice bearing syngeneic or xenograft tumors was associated with regressions that were comparable with those found when dosing on a daily basis with GO-203. Conclusions: These findings thus define an effective approach for (i) sustained administration of GO-203 in polymeric PLA-(PEG-PPG-PEG) nanoparticles to target MUC1-C in cancer cells and (ii) the potential delivery of other anticancer peptide drugs. Clin Cancer Res; 21(10); 2338–47. ©2015 AACR.