Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

University of Calgary

Traumatic peripheral nerve injuries are common and often result in partial or permanent paralysis, numbness of the affected limb, and debilitating neuropathic pain. Experimental animal models of nerve injury have utilized a diversity of outcome measures to examine functional recovery following injury. Four primary categories of outcome measures of regenerative success including retrograde labeling with counts of regenerating neurons, immunohistochemistry and histomorphometry, reinnervation of target muscles, and behavioral analysis of recovery will be reviewed. Validity of different outcome measures are discussed in context of hindlimb, forelimb, and facial nerve injury models. Severity of nerve injury will be highlighted, and comparisons between nerve crush injury and more severe transection and neuroma-in-continuity nerve injury paradigms will be evaluated. The case is made that specific outcome measures may be more sensitive to assessing functional recovery following nerve injury than others. This will be discussed in the context of the lack of association between certain outcome measures of nerve regeneration. Examples of inaccurate conclusions from specific outcome measures will also be considered. Overall, researchers must therefore take care to select appropriate outcome measures for animal nerve injury studies dependant on the specific experimental interventions and scientific questions addressed.

Comparative outcome measures in peripheral regeneration studies.

Pro-regenerative macrophages are well known for their role in promoting tissue repair; however, their specific roles in promoting regeneration of the injured nerve are not well defined. Specifically, how macrophages interact with Schwann cells following injury during remyelination has been largely unexplored. We demonstrate that after injury, including in humans, macrophages function to clear debris and persist within the nerve microenvironment. Macrophage ablation immediately preceding remyelination results in an increase in immature Schwann cell density, a reduction in remyelination, and long-term deficits in conduction velocity. Targeted RNA-seq of macrophages from injured nerve identified Gas6 as one of several candidate factors involved in regulating Schwann cell dynamics. Functional studies show that the absence of Gas6 within monocyte lineage cells impairs Schwann cell remyelination within the injured nerve. These results demonstrate a role for macrophages in regulating Schwann cell function during nerve regeneration and highlight a molecular mechanism by which this occurs.

Macrophages Regulate Schwann Cell Maturation after Nerve Injury.

 Deciphering avenues to adequately control malignancies in the peripheral nerve will reduce the need for current, largely-ineffective, standards of care which includes the use of invasive, nerve-damaging, resection surgery. By avoiding the need for en bloc resection surgery, the likelihood of retained function or efficient nerve regeneration following the control of tumor growth is greater, which has several implications for long-term health and well-being of cancer survivors. Nerve tumors can arise as malignant peripheral nerve sheath tumors (MPNST) that result in a highly-aggressive form of soft tissue sarcoma. Although the precise cause of MPNST remains unknown, studies suggest that dysregulation of Schwann cells, mediated by the microenvironment, plays a key role in tumor progression. This study aimed to further characterize the role of local microenvironment on tumor progression, with an emphasis on identifying factors within tumor suppressive environments that have potential for therapeutic application.  We created GFP-tagged adult induced tumorigenic Schwann cell lines (iSCs) and transplanted them into various  microenvironments. We used immunohistochemistry to document the response of iSCs and performed proteomics analysis to identify local factors that might modulate divergent iSC behaviors.  Following transplant into the skin, spinal cord or epineurial compartment of the nerve, iSCs formed tumors closely resembling MPNST. In contrast, transplantation into the endoneurial compartment of the nerve significantly suppressed iSC proliferation. Proteomics analysis revealed a battery of factors enriched within the endoneurial compartment, of which one growth factor of interest, ciliary neurotrophic factor (CNTF) was capable of preventing iSCs proliferation .  This dataset describes a novel approach for identifying biologically relevant therapeutic targets, such as CNTF, and highlights the complex relationship that tumor cells have with their local microenvironment. This study has significant implications for the development of future therapeutic strategies to fight MPNSTs, and, consequently, improve peripheral nerve regeneration and nerve function.

Rajiv Midha

Hotchkiss Brain Institute,
Department of Clinical Neurosciences,
Cumming School of Medicine,
Department of Clinical Neurosciences, Cumming School of Medicine

University of Calgary

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

Rajiv Midha

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Hotchkiss Brain Institute,Department of Clinical Neurosciences,Cumming School of Medicine,Department of Clinical Neurosciences, Cumming School of Medicine

University of Calgary

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

Hotchkiss Brain Institute,
Department of Clinical Neurosciences,
Cumming School of Medicine,
Department of Clinical Neurosciences, Cumming School of Medicine