skeletal muscle cells preparation for loading into mass cytometer and cytof data analysis

Single-Cell CyTOF Analysis of Myogenic Progenitors in Muscle Regeneration

Skeletal muscle constitutes the largest tissue by mass in the body and regulates multiple functions, from eyesight to respiration, from posture to movement, as well as metabolism1. Therefore, maintaining skeletal muscle integrity and function is critical to health. Skeletal muscle tissue, which consists of tightly packed bundles of multinucleated myofibers surrounded by a complex network of nerves and blood vessels, exhibits remarkable regenerative potential1,2.
The main drivers of skeletal muscle regeneration are adult muscle stem cells (MuSCs). Also known as satellite cells, due to their unique anatomical location adjacent to the plasma membrane of the myofiber and beneath the basal lamina, they were first identified in 19613. MuSCs express a unique molecular marker, the transcription factor paired box 7 (Pax7)4. Mostly quiescent in healthy adults, they become activated upon muscle injury and proliferate to give rise to progeny that will (i) differentiate into fusion-competent muscle cells that will form new myofibers to repair muscle damage or (ii) self-renew to replenish the stem cell pool5.
At the cellular and molecular level, the process of regeneration is quite dynamic and involves cell-state transitions, characterized by the coordinated expression of key myogenic transcription factors, also known as myogenic regulatory factors (MRFs)6,7. Prior in vivo developmental studies, lineage tracing experiments, and cell culture work using myoblasts&#160;have shown that sequential expression of these transcription factors drives myogenesis, with myogenic factor 5 (Myf5) being expressed upon activation, myogenic differentiation 1 (MyoD1) expression marking commitment to the myogenic program, and myogenin (MyoG) expression marking differentiation8,9,10,11,12,13,14. Despite this knowledge and the discovery of cell surface markers to purify MuSCs, strategies and tools to identify and isolate discrete populations along the myogenic differentiation path and resolve a myogenic progression in vivo have been lacking15,16,17,18.
Here, we present a novel method, based on recently published research, which enables the identification of stem and progenitor cells in skeletal muscle and the analysis of their cellular, molecular, and proliferation dynamics in the context of acute muscle injury19. This approach relies on single-cell mass cytometry (also known as Cytometry by Time of Flight [CyTOF]) to simultaneously detect key cell surface markers (&#945;7&#160;integrin, CD9, CD44, CD98, and CD104), intracellular myogenic transcription factors (Pax7, Myf5, MyoD, and MyoG) and a nucleoside analog (5-Iodo-2&#8242;-deoxyuridine, IdU), to monitor cells in S phase19,20,21,22,23. Moreover, the protocol presents a strategy based on the detection of two cell surface markers, CD9 and CD104, to purify these cell populations by fluorescence-activated cell sorting (FACS), therefore enabling future in-depth studies of their function in the context of injury and muscle diseases. While primary myoblasts have been extensively used in the past to study the late stages of myogenic differentiation in vitro, it is not known whether they recapitulate the molecular state of muscle progenitor cells found in vivo24,25,26,27,28,29,30. The production of myoblasts is laborious and time-consuming, and the molecular state of this primary culture changes rapidly upon passaging31. Hence, freshly isolated myogenic progenitors purified with this method will provide a more physiological system to study myogenesis and the effect of genetic or pharmacological manipulations ex-vivo.
The protocol presented here can be applied to address a variety of research questions, for example, to study the dynamics of the myogenic compartment in vivo in animal models of muscle diseases, in response to acute genetic manipulations or upon pharmacological interventions, therefore deepening our understanding of muscle stem cell dysfunction in different biological contexts and facilitating the development of novel therapeutic interventions.

Author Spotlight: Investigating Cellular and Molecular Dynamics During Muscle Regeneration Using Cutting-Edge Single-Cell Technologies

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

Our research investigates the molecular mechanisms underlying stem cell dysfunction in aging and disease. Our goal is to understand how muscle stem and progenitor cells interact with other cells in the muscle microenvironment to achieve effective regeneration. We are also studying whether a barren cell communication impacts muscle stem cell function in aging and disease.A major barrier to investigating cell-cell communication within the stem cell niche has been the lack of technologies that provide single cell resolution. Single cell mass cytometry allows simultaneous high throughput quantitative analysis on multiple cell types and molecular phenotypes in complex tissues. Our protocol enables purification and deep profiling of rare stem cells and the progeny during muscle regeneration using antibodies to cell surface markers and myogenic transcription factors.Studying these cells helps us understand how muscle stem cell fates is controlled, and uncover the networks that drive cell state transitions doing effective regeneration. Measuring up to 50 cell surface and intracellular protein simultaneously at a single cell level is a valuable approach for investigating cellular function in muscle tissue. This technique can identify unique molecular signatures and reveal novel mechanisms of muscle stem cell dysfunction in aging and disease.This protocol allows to investigate how cell state transition impact tissue regeneration in health and disease. The signature of activated stem cells we present here enable studies of stem cell quiescence. They were not previously possible.We can now purify activated stem cells, CD98 High, CD44 High, based solely on cell surface marker, and study for the first time, how they return to quiescence.

Skeletal Muscle Cells Preparation for Loading into Mass Cytometer and CyTOF Data Analysis

To begin, take the skeletal muscle cells stained with cisplatin, metal conjugated antibodies, and intercalator-iridium solution, and pellet the cells by centrifugation. Once the supernatant is removed, add one milliliter of CSM to the vortexed cells. Pellet the cells by centrifugation and wash in one milliliter of CAS buffer.After centrifugation, aspirate the supernatant to approximately 200 microliters. Add one milliliter of CAS buffer to the vortexed samples and aliquot five microliters for cell count. After centrifuging the cells and aspirating the supernatant to 50 microliters, resuspend the cells in CAS buffer to a final concentration of 1 million cells per milliliter and add calibration beads to achieve a final concentration of 0.1x.Load the sample into the mass cytometer to collect data using a flow rate of 400 to 500 cells per second, and normalize the data after collection. If necessary, concatenate individual Flow Cytometry Standard or FCS files for each sample into a single file. Identify single cells by gating on iridium intercalator positive events.Select events that are negative for cisplatin to gate for live cells. Gate on the population of interest and quantify the relative proportion of stem and progenitor cells. To perform high-dimensional analysis, export the population of interest and use clustering algorithms.For X-shift analysis, download the Vortex software package and Java 64-bit. Upload the exported cell populations to a local database and define the clustering parameters. To visualize the spatial relationships between cell populations within the X-shift clusters, perform a force-directed layout.CyTOF analysis identified a sequence of four populations, stem cells, and progenitor populations one through three. The progenitor populations P1 and P2 correspond to increasingly more mature progenitor cells. P3 was previously identified but not characterized due to its low abundance.The stem and progenitor cell dynamics post-injury were analyzed using CD9 by CD104 by axial dot plots. A notable increase in the stem cell population was observed on day three, suggesting expansion. This was supported by increased iododeoxyuridine incorporation, indicating cell proliferation, and heightened MyoD expression as shown by the color overlay.Activated stem cells marked by high levels of CD98, CD44, MyoD, and iododeoxyuridine were identified, underscoring their high proliferation and activated state at day three post-injury.

skeletal-muscle-cells-preparation-for-loading-into-mass-cytometer

Research

JoVE Journal

Biology

32 Views. To begin, take the skeletal muscle cells stained with cisplatin, metal conjugated antibodies, and intercalator-iridium solution, and pellet the cells by centrifugation. Once the supernatant is removed, add one milliliter of CSM to the vortexed cells. Pellet the cells by centrifugation and wash in one milliliter of CAS buffer.After centrifugation, aspirate the supernatant to approximately 200 microliters. Add one milliliter of CAS buffer to the vortexed samples and aliquot five microlit...