Under stress conditions such as infection or inflammation the body rapidly needs to generate new blood cells that are adapted to the challenge. Haematopoietic cytokines are known to increase output of specific mature cells by affecting survival, expansion and differentiation of lineage-committed progenitors1,2, but it has been debated whether long-term haematopoietic stem cells (HSCs) are susceptible to direct lineage-specifying effects of cytokines. Although genetic changes in transcription factor balance can sensitize HSCs to cytokine instruction3, the initiation of HSC commitment is generally thought to be triggered by stochastic fluctuation in cell-intrinsic regulators such as lineage-specific transcription factors4–7, leaving cytokines to ensure survival and proliferation of the progeny cells8,9. Here we show that macrophage colony-stimulating factor (M-CSF, also called CSF1), a myeloid cytokine released during infection and inflammation, can directly induce the myeloid master regulator PU.1 and instruct myeloid cell-fate change in mouse HSCs, independently of selective survival or proliferation. Video imaging and single-cell gene expression analysis revealed that stimulation of highly purified HSCs with M-CSF in culture resulted in activation of the PU.1 promoter and an increased number of PU.1+ cells with myeloid gene signature and differentiation potential. In vivo, high systemic levels of M-CSF directly stimulated M-CSF-receptor-dependent activation of endogenous PU.1 protein in single HSCs and induced a PU.1-dependent myeloid differentiation preference. Our data demonstrate that lineage-specific cytokines can act directly on HSCs in vitro and in vivo to instruct a change of cell identity. This fundamentally changes the current view of how HSCs respond to environmental challenge and implicates stress-induced cytokines as direct instructors of HSC fate.
Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency)1–6. In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing7,8. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection9,10. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods11,12. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.
Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke.
Gene/oligonucleotide therapies have emerged as a promising strategy for the treatment of different neurological conditions. However, current methodologies for the delivery of neurogenic/neurotrophic cargo to brain and nerve tissue are fraught with caveats, including reliance on viral vectors, potential toxicity, and immune/inflammatory responses. Moreover, delivery to the central nervous system is further compounded by the low permeability of the blood brain barrier. Extracellular vesicles (EVs) have emerged as promising delivery vehicles for neurogenic/neurotrophic therapies, overcoming many of the limitations mentioned above. However, the manufacturing processes used for therapeutic EVs remain poorly understood. Here, we conducted a detailed study of the manufacturing process of neurogenic EVs by characterizing the nature of cargo and surface decoration, as well as the transfer dynamics across donor cells, EVs, and recipient cells. Neurogenic EVs loaded with Ascl1, Brn2, and Myt1l (ABM) are found to show enhanced neuron-specific tropism, modulate electrophysiological activity in neuronal cultures, and drive pro-neurogenic conversions/reprogramming. Moreover, murine studies demonstrate that surface decoration with glutamate receptors appears to mediate enhanced EV delivery to the brain. Altogether, the results indicate that ABM-loaded designer EVs can be a promising platform nanotechnology to drive pro-neuronal responses, and that surface functionalization with glutamate receptors can facilitate the deployment of EVs to the brain.
Extracellular vesicles (EVs) have emerged as a promising carrier system for the delivery of therapeutic payloads in multiple disease models, including cancer. However, effective targeting of EVs to cancerous tissue remains a challenge. Here, it is shown that nonviral transfection of myeloid-derived suppressor cells (MDSCs) can be leveraged to drive targeted release of engineered EVs that can modulate transfer and overexpression of therapeutic anticancer genes in tumor cells and tissue. MDSCs are immature immune cells that exhibit enhanced tropism toward tumor tissue and play a role in modulating tumor progression. Current MDSC research has been mostly focused on mitigating immunosuppression in the tumor niche; however, the tumor homing abilities of these cells present untapped potential to deliver EV therapeutics directly to cancerous tissue. In vivo and ex vivo studies with murine models of breast cancer show that nonviral transfection of MDSCs does not hinder their ability to home to cancerous tissue. Moreover, transfected MDSCs can release engineered EVs and mediate antitumoral responses via paracrine signaling, including decreased invasion/metastatic activity and increased apoptosis/necrosis. Altogether, these findings indicate that MDSCs can be a powerful tool for the deployment of EV-based therapeutics to tumor tissue.
Myeloid-derived suppressor cells (MDScs) are immune cells that exert immunosuppression within the tumor, protecting cancer cells from the host's immune system and/or exogenous immunotherapies.While current research has been mostly focused in countering MDSC-driven immunosuppression, little is known about the mechanisms by which MDSCs disseminate/infiltrate cancerous tissue. This study looks into the use of microtextured surfaces, coupled with in vitro and in vivo cellular and molecular analysis tools, to videoscopically evaluate the dissemination patterns of MDSCs under structurally guided migration, at the single-cell level. MDSCs exhibited topographically driven migration, showing significant intra-and inter-population differences in motility, with velocities reaching ~40 μm h −1 . Downstream analyses coupled with single-cell migration uncovered the presence of specific MDSC subpopulations with different degrees of tumor-infiltrating and anti-inflammatory capabilities. Granulocytic MDSCs showed a ~≥3-fold increase in maximum dissemination velocities and traveled distances, and a ~10-fold difference in the expression of pro-and anti-inflammatory markers. Prolonged culture also revealed that purified subpopulations of MDSCs exhibit remarkable plasticity, with homogeneous/sorted subpopulations giving rise to heterogenous cultures that represented the entire hierarchy of MDSC phenotypes within 7 days. These studies point towards the granulocytic subtype as a potential cellular target of interest given their superior dissemination ability and enhanced antiinflammatory activity.The tumor microenvironment is highly heterogeneous in nature, with cancerous cells co-habiting with both stromal and immune cells. Such complex cellular interplay plays a central role in modulating tumor progression. Myeloid-derived suppressor cells (MDSCs), in particular, have been known to exert immunosuppressive activity in the tumor niche, which protects cancerous cells from the host immune system and/or different therapeutic modalities 1,2 . While a lot of research has been devoted to developing advanced drugs and drug delivery systems to target cancerous cells 3-5 , and/or blocking MDSC-driven immunosuppression within the tumor niche 6,7 , less is known about the motility mechanisms by which MDSCs disseminate and colonize the tumor in the first place.MDSCs are innate immune cells that are highly expanded in cancer patients 2 . These cells tend to infiltrate tumors and lymphoid tissues, and their levels correlate with increased tumor burden and limited survival in a variety of malignancies 6-9 . MDSCs specifically contribute to the loss of immune effector cell function and reduce the efficacy of immunotherapies. As such, MDSCs have emerged as an attractive therapeutic target in
Myeloid derived suppressor cells (MDSCs) have gained significant attention for their immunosuppressive role in cancer and their ability to contribute to tumor progression and metastasis. Understanding the role of MDSCs in driving cancer cell migration, a process fundamental to metastasis, is essential to fully comprehend and target MDSC‐tumor cell interactions. This study employs microfabricated platforms, which simulate the structural cues present in the tumor microenvironment (TME) to elucidate the effects of MDSCs on the migratory phenotype of cancer cells at the single cell level. The results indicate that the presence of MDSCs enhances the motility of cancer‐epithelial cells when directional cues (either topographical or spatial) are present. This behavior appears to be independent of cell–cell contact and driven by soluble byproducts from heterotypic interactions between MDSCs and cancer cells. Moreover, MDSC cell‐motility is also impacted by the presence of cancer cells and the cancer cell secretome in the presence of directional cues. Epithelial dedifferentiation is the likely mechanism for changes in cancer cell motility in response to MDSCs. These results highlight the biochemical and biostructural conditions under which MDSCs can support cancer cell migration, and could therefore provide new avenues of research and therapy aimed at stemming cancer progression.
While gene and cell therapies have emerged as promising treatment strategies for various neurological conditions, heavy reliance on viral vectors can hamper widespread clinical implementation. Here, the use of tissue nanotransfection as a platform nanotechnology to drive nonviral gene delivery to nerve tissue via nanochannels, in an effective, controlled, and benign manner is explored. TNT facilitates plasmid DNA delivery to the sciatic nerve of mice in a voltage‐dependent manner. Compared to standard bulk electroporation (BEP), impairment in toe‐spread and pinprick response is not caused by TNT, and has limited to no impact on electrophysiological parameters. BEP, however, induces significant nerve damage and increases macrophage immunoreactivity. TNT is subsequently used to deliver vasculogenic cell therapies to crushed nerves via delivery of reprogramming factor genes Etv2, Foxc2, and Fli1 (EFF). The results indicate the TNT‐based delivery of EFF in a sciatic nerve crush model leads to increased vascularity, reduced macrophage infiltration, and improved recovery in electrophysiological parameters compared to crushed nerves that are TNT‐treated with sham/empty plasmids. Altogether, the results indicate that TNT can be a powerful platform nanotechnology for localized nonviral gene delivery to nerve tissue, in vivo, and the deployment of reprogramming‐based cell therapies for nerve repair/regeneration.
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