Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids.

Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5 - /KRT17 + aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.

The Efficacy of Schwann-Like Differentiated Muscle-Derived Stem Cells in Treating Rodent Upper Extremity Peripheral Nerve Injury.

BACKGROUND: There is a pressing need to identify alternative mesenchymal stem cell sources for Schwann cell cellular replacement therapy, to improve peripheral nerve regeneration. This study assessed the efficacy of Schwann cell-like cells (induced muscle-derived stem cells) differentiated from muscle-derived stem cells (MDSCs) in augmenting nerve regeneration and improving muscle function after nerve trauma. METHODS: The Schwann cell-like nature of induced MDSCs was characterized in vitro using immunofluorescence, flow cytometry, microarray, and reverse-transcription polymerase chain reaction. In vivo, four groups (n = 5 per group) of rats with median nerve injuries were examined: group 1 animals were treated with intraneural phosphate-buffered saline after cold and crush axonotmesis (negative control); group 2 animals were no-injury controls; group 3 animals were treated with intraneural green fluorescent protein-positive MDSCs; and group 4 animals were treated with green fluorescent protein-positive induced MDSCs. All animals underwent weekly upper extremity functional testing. Rats were euthanized 5 weeks after treatment. The median nerve and extrinsic finger flexors were harvested for nerve histomorphometry, myelination, muscle weight, and atrophy analyses. RESULTS: In vitro, induced MDSCs recapitulated native Schwann cell gene expression patterns and up-regulated pathways involved in neuronal growth/signaling. In vivo, green fluorescent protein-positive induced MDSCs remained stably transformed 5 weeks after injection. Induced MDSC therapy decreased muscle atrophy after median nerve injury (p = 0.0143). Induced MDSC- and MDSC-treated animals demonstrated greater functional muscle recovery when compared to untreated controls (hand grip after induced MDSC treatment: group 1, 0.91 N; group 4, 3.38 N); p < 0.0001) at 5 weeks after treatment. This may demonstrate the potential beneficial effects of MDSC therapy, regardless of differentiation stage. CONCLUSION: Both MDSCs and induced MDSCs decrease denervation muscle atrophy and improve subsequent functional outcomes after upper extremity nerve trauma in rodents.

Oligodendrocyte precursors gain Schwann cell-like phenotype and remyelinate axons upon engraftment into peripheral nerves.

The ability to treat large peripheral nerve injuries may be greatly advanced if an accessible source of human myelinating cells is identified, as it overcomes one of the major limitations of acellular or synthetic nerve guides compared with autografts, the gold standard for large defect repair. Methods to derive oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells have advanced to the point where they have been shown capable of myelination and are being evaluated in clinical trials. Here, we test the hypothesis that OPCs can survive and remyelinate axons in the peripheral nervous system during a repair process. Using freshly isolated OPCs from mouse post-natal brains, we engrafted these OPCs into the tibial nerve immediately after it being subjected to cryolesioning. At 1-month postengraftment, we found numerous graft-derived cells that survived in this environment, and many transplanted cells expressed Schwann cell markers such as periaxin and S100ß coexpressed with myelin basic protein, whereas oligodendrocyte markers O4 and Olig2 were virtually absent. Our results demonstrate that OPCs can survive in a peripheral nervous system micro-environment and undergo niche-dependent transdifferentiation into Schwann cell-like cells as has previously been observed in central nervous system focal demyelination models, suggesting that OPCs constitute an accessible source of cells for peripheral nerve cell therapies.

Egr2 overexpression in Schwann cells increases myelination frequency in vitro.

Schwann cells are key players in peripheral nerve regeneration, and are uniquely capable of remyelinating axons in this context. Schwann cells orchestrate this process via a set of transcription factors. While it has been shown that overexpression of specific genes, e.g. Egr2, upregulates myelin-related transcripts, it remains unknown if such manipulation can functionalize the cells and enhance their myelination frequency. The ability to do so could have implications in the use of human stem cell-derived Schwann cells, where myelination is hard to achieve. After screening four candidate transcription factors (Sox10, Oct6, Brn2 and Egr2), we found that overexpression of Egr2 in rat Schwann cells co-cultured with sensory neurons enhanced myelination frequency and reduced cell proliferation. However, in a mouse model of sciatic nerve repair with cells engrafted within a nerve guide, myelination frequency in the engrafted cells was reduced upon Egr2 overexpression. Our results show that while overexpression of Egr2 can enhance the myelination frequency in vitro, it is context-dependent, potentially influenced by the microenvironment, timing of association with axons, expression level, species differences, or other factors.

Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy.

Stem cell-based approaches to improve nerve regeneration: potential implications for reconstructive transplantation?

Reconstructive transplantation has become a viable option to restore form and function after devastating tissue loss. Functional recovery is a key determinant of overall success and critically depends on the quality and pace of nerve regeneration. Several molecular and cell-based therapies have been postulated and tested in pre-clinical animal models to enhance nerve regeneration. Schwann cells remain the mainstay of research focus providing neurotrophic support and signaling cues for regenerating axons. Alternative cell sources such as mesenchymal stem cells and adipose-derived stromal cells have also been tested in pre-clinical animal models and in clinical trials due to their relative ease of harvest, rapid expansion in vitro, minimal immunogenicity, and capacity to integrate and survive within host tissues, thereby overcoming many of the challenges faced by culturing of human Schwann cells and nerve allografting. Induced pluripotent stem cell-derived Schwann cells are of particular interest since they can provide abundant, patient-specific autologous Schwann cells. The majority of experimental evidence on cell-based therapies, however, has been generated using stem cell-seeded nerve guides that were developed to enhance nerve regeneration across "gaps" in neural repair. Although primary end-to-end repair is the preferred method of neurorrhaphy in reconstructive transplantation, mechanistic studies elucidating the principles of cell-based therapies from nerve guidance conduits will form the foundation of further research employing stem cells in end-to-end repair of donor and recipient nerves. This review presents key components of nerve regeneration in reconstructive transplantation and highlights the pre-clinical studies that utilize stem cells to enhance nerve regeneration.

Signaling cue presentation and cell delivery to promote nerve regeneration.

Limitations in current nerve regeneration techniques have stimulated the development of various approaches to mimic the extrinsic cues available in the natural nerve regeneration environment. Biomaterials approaches modulate the microenvironment of a regenerating nerve through tailored presentation of signaling molecules, creating physical and biochemical guidance cues to direct axonal regrowth across nerve lesion sites. Cell-based approaches center on increasing the neurotrophic support, adhesion guidance and myelination capacity of Schwann cells and other alternative cell types to enhance nerve regrowth and functional recovery. Recent advances in presenting directional guidance cues in nerve guidance conduits and improving the regenerative outcomes of cell delivery provide inspirations to engineering the next generation of nerve repair solutions.