HybriSeq: Probe-based Device-free Single-cell RNA Profiling.

We have developed the HybriSeq method for single-cell RNA profiling, which utilizes in situ hybridization of multiple probes for targeted transcripts, followed by split-pool barcoding and sequencing analysis of the probes. We have shown that HybriSeq can achieve high sensitivity for RNA detection with multiple probes and profile RNA accessibility. The utility of HybriSeq is demonstrated in characterizing cell-to-cell heterogeneities of a panel of 95 cell-cycle-related genes and the probe-probe heterogeneity within a single transcript.

The Combination of Synoeca-MP Antimicrobial Peptide with IDR-1018 Stimulates Proliferation, Migration, and the Expression of Pro-Regenerative Genes in Both Human Skin Cell Cultures and 3D Skin Equivalents.

In skin lesions, the development of microbial infection affects the healing process, increasing morbidity and mortality rates in patients with severe burns, diabetic foot, and other types of skin injuries. Synoeca-MP is an antimicrobial peptide (AMP) that exhibits activity against several bacteria of clinical importance, but its cytotoxicity can represent a problem for its positioning as an effective antimicrobial compound. In contrast, the immunomodulatory peptide IDR-1018 presents low toxicity and a wide regenerative potential due to its ability to reduce apoptotic mRNA expression and promote skin cell proliferation. In the present study, we used human skin cells and a 3D skin equivalent models to analyze the potential of the IDR-1018 peptide to attenuate the cytotoxicity of synoeca-MP, as well as the influence of synoeca-MP/IDR-1018 combination on cell proliferation, regenerative processes, and wound repair. We found that the addition of IDR-1018 significantly improved the biological properties of synoeca-MP on skin cells without modifying its antibacterial activity against S. aureus. Likewise, in both melanocytes and keratinocytes, the treatment with synoeca-MP/IDR-1018 combination induces cell proliferation and migration, while in a 3D human skin equivalent model, it can accelerate wound reepithelization. Furthermore, treatment with this peptide combination generates an up-regulation in the expression of pro-regenerative genes in both monolayer cell cultures and in 3D skin equivalents. This data suggests that the synoeca-MP/IDR-1018 combination possesses a good profile of antimicrobial and pro-regenerative activity, opening the door to the development of new strategies for the treatment of skin lesions.

Senotherapeutic peptide treatment reduces biological age and senescence burden in human skin models.

Cellular senescence is known to play a role in age-related skin function deterioration which potentially influences longevity. Here, a two-step phenotypic screening was performed to identify senotherapeutic peptides, leading to the identification of Peptide (Pep) 14. Pep 14 effectively decreased human dermal fibroblast senescence burden induced by Hutchinson-Gilford Progeria Syndrome (HGPS), chronological aging, ultraviolet-B radiation (UVB), and etoposide treatment, without inducing significant toxicity. Pep 14 functions via modulation of PP2A, an understudied holoenzyme that promotes genomic stability and is involved in DNA repair and senescence pathways. At the single-cell level, Pep 14 modulates genes that prevent senescence progression by arresting the cell cycle and enhancing DNA repair, which consequently reduce the number of cells progressing to late senescence. When applied on aged ex vivo skin, Pep 14 promoted a healthy skin phenotype with structural and molecular resemblance to young ex vivo skin, decreased the expression of senescence markers, including SASP, and reduced the DNA methylation age. In summary, this work shows the safe reduction of the biological age of ex vivo human skins by a senomorphic peptide.

In vitro and in vivo toxicity assessment of the senotherapeutic Peptide 14.

Senotherapeutic molecules decrease cellular senescence burden, constituting promising approaches to combat the accumulation of senescent cells observed in chronological aging and age-related diseases. Numerous molecules have displayed senotherapeutic potential, but toxicity has been frequently observed. Recently, a new senotherapeutic compound, Peptide 14, was developed to modulate cellular senescence in the skin. In order to assess the potential toxic and genotoxic effects of the peptide, we observed the viability of human primary dermal fibroblasts and epidermal keratinocytes with Peptide 14 treatment, and show that it is mostly non-toxic in concentrations up to 100 µM. Cancer lines were also used to investigate its potential of modulating proliferation. Different concentrations of the peptide promoted a discrete reduction in the proliferation of cancerous cells of the MeWo and HeLa lineages. In full-thickness human skin equivalents, topically formulated Peptide 14 also failed to exert any significant irritation, nor cellular toxicity when added to the culture media. Genotoxic assays including the Ames, micronucleus, and karyotyping tests also indicate the safety of the peptide. Finally, the irritative potential of the peptide was assessed in human subjects in a repeated insult patch test executed using 1 mM peptide. No visible skin reactions were observed in any of the 54 participants. Taken together, the present data support that Peptide 14 is a senotherapeutic molecule with a positive safety profile as tested with cruelty-free models, justifying further studies involving the peptide.

IDR-1018 induces cell proliferation, migration, and reparative gene expression in 2D culture and 3D human skin equivalents.

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR-1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR-1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR-1018 using human 2D and 3D human skin equivalents. First, we investigated IDR-1018 using human cells found in skin-primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR-1018 promoted cell proliferation and expression of marker of proliferation Ki-67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki-67, matrix metalloproteinase 1, C-X-C motif chemokine receptor type 4, C-X-C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR-1018 treatment. The capacity of IDR-1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR-1018-treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR-1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.

Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation.

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-ß1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-ß may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-ß3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.

Differential Regulation of Human Bone Marrow Mesenchymal Stromal Cell Chondrogenesis by Hypoxia Inducible Factor-1α Hydroxylase Inhibitors.

The transcriptional profile induced by hypoxia plays important roles in the chondrogenic differentiation of marrow stromal/stem cells (MSC) and is mediated by the hypoxia inducible factor (HIF) complex. However, various compounds can also stabilize HIF's oxygen-responsive element, HIF-1α, at normoxia and mimic many hypoxia-induced cellular responses. Such compounds may prove efficacious in cartilage tissue engineering, where microenvironmental cues may mediate functional tissue formation. Here, we investigated three HIF-stabilizing compounds, which each have distinct mechanisms of action, to understand how they differentially influenced the chondrogenesis of human bone marrow-derived MSC (hBM-MSC) in vitro. hBM-MSCs were chondrogenically-induced in transforming growth factor-ß3-containing media in the presence of HIF-stabilizing compounds. HIF-1α stabilization was assessed by HIF-1α immunofluorescence staining, expression of HIF target and articular chondrocyte specific genes by quantitative polymerase chain reaction, and cartilage-like extracellular matrix production by immunofluorescence and histochemical staining. We demonstrate that all three compounds induced similar levels of HIF-1α nuclear localization. However, while the 2-oxoglutarate analog dimethyloxalylglycine (DMOG) promoted upregulation of a selection of HIF target genes, desferrioxamine (DFX) and cobalt chloride (CoCl2 ), compounds that chelate or compete with divalent iron (Fe2+ ), respectively, did not. Moreover, DMOG induced a more chondrogenic transcriptional profile, which was abolished by Acriflavine, an inhibitor of HIF-1α-HIF-ß binding, while the chondrogenic effects of DFX and CoCl2 were more limited. Together, these data suggest that HIF-1α function during hBM-MSC chondrogenesis may be regulated by mechanisms with a greater dependence on 2-oxoglutarate than Fe2+ availability. These results may have important implications for understanding cartilage disease and developing targeted therapies for cartilage repair. Stem Cells 2018;36:1380-1392.

Exploiting Advanced Hydrogel Technologies to Address Key Challenges in Regenerative Medicine.

Regenerative medicine aims to tackle a panoply of challenges from repairing focal damage to articular cartilage to preventing pathological tissue remodeling after myocardial infarction. Hydrogels are water-swollen networks formed from synthetic or naturally derived polymers and are emerging as important tools to address these challenges. Recent advances in hydrogel chemistries are enabling researchers to create hydrogels that can act as 3D ex vivo tissue models, allowing them to explore fundamental questions in cell biology by replicating tissues' dynamic and nonlinear physical properties. Enabled by cutting edge techniques such as 3D bioprinting, cell-laden hydrogels are also being developed with highly controlled tissue-specific architectures, vasculature, and biological functions that together can direct tissue repair. Moreover, advanced in situ forming and acellular hydrogels are increasingly finding use as delivery vehicles for bioactive compounds and in mediating host cell response. Here, advances in the design and fabrication of hydrogels for regenerative medicine are reviewed. It is also addressed how controlled chemistries are allowing for precise engineering of spatial and time-dependent properties in hydrogels with a look to how these materials will eventually translate to clinical applications.