pulverization and homogenization of frozen human intermuscular adipose tissue samples for nuclei isolation

Isolierung von Einzelzellkernen aus IMAT zur Untersuchung von Genexpressionsprofilen 

Intermuscular adipose tissue (IMAT) is an ectopic adipose depot residing between and around muscle fibers1. As described in detail in a recent review by Goodpaster et al., IMAT can be detected using high-resolution computed tomography (CT) and magnetic resonance imaging (MRI) (Figure 1A,B) and is found around and within muscle fibers throughout the entire body1. The quantity of IMAT varies greatly between individuals and is influenced by BMI, age, sex, race, and sedentariness2,3,4. Moreover, IMAT deposition is commonly seen in pathological conditions associated with muscle degeneration5, and numerous studies have documented increased IMAT mass in individuals with obesity, type 2 diabetes, metabolic syndrome, and insulin resistance6,7,8,9. Nonetheless, the cellular and biological properties of IMAT are only beginning to be unraveled. The limited accessibility and the variation in IMAT locations and content throughout the body have challenged the collection of samples from this unique adipose depot2. Moreover, samples are easily &#39;contaminated&#39; with skeletal muscle (SM) upon collection, making the separation between the biological contribution from the different tissues difficult to decipher (Figure 1C). To this end, single nuclei RNA sequencing (snRNA-seq), which has gained considerable attention during the last decade, serves as an ideal methodology to allow for the separation of IMAT- and SM-derived gene expression patterns with single-cell resolution. Moreover, nuclei isolation is particularly useful for adipose tissue due to the large lipid-laden adipocytes, which are impossible to dissociate into single-cell suspension without compromising the integrity of the cells. Lastly, this technology holds the potential to discover novel markers of IMAT-specific adipocytes and uncover the composition and presence of different progenitor cell populations, as well as study the variation of the cell composition in pathological and normal conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66784/66784fig01.jpg" /> 
Figure 1: Images of IMAT. Representative magnetic resonance (MRI) image of IMAT from (A) a middle-aged lean female and (B) a middle-aged male with obesity. Red: subcutaneous adipose tissue, yellow: intermuscular adipose tissue, green: skeletal muscle, blue: bone. Image courtesy of Heather Cornnell, AdventHealth Translational Research Institute. (C) Fresh tissue sample with IMAT (encircled by dashed black line). Image courtesy of Meghan Hopf, AdventHealth Translational Research Institute and Bryan Bergman, University of Colorado. This figure has been modified with permission from Goodpaster et al.1. <a href="https://www.jove.com/files/ftp_upload/66784/66784fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A number of studies have been published from the livestock industry investigating the marbling of meat (IMAT in particular) in pigs, chickens, and cattle using single-cell (sc) and snRNA-seq10. These studies have identified several subpopulations of adipocytes and markers of potential progenitor cells of IMAT11,12,13; however, whether these cellular compositions translate to human IMAT is unknown. To our knowledge, only one study has looked into the cellular heterogeneity of human muscle with fatty infiltration, obtained from male patients with hip osteoarthritis, using snRNA-seq14. The investigators reported a small adipocyte population and several fibro-adipogenic progenitor (FAP) subpopulations within the large population of myonuclei14. Our study is the first to develop a method to directly interrogate IMAT manually dissected&#160;from human muscle for cellular composition using snRNA-seq.
Importantly, protocols for snRNA-seq need to be customized for the specific tissue studied, as the amount of tissue available and the physical properties of the specific tissue will dictate the optimal processing steps. The tissue yield for IMAT is typically small, often not exceeding 50 mg, even when performing ultrasound-guided biopsies. Hence, proper processing of this scarce tissue is essential. We believe that this protocol will serve as a valuable resource for researchers studying human IMAT.

Autor im Rampenlicht: Entschlüsselung der zellulären Geheimnisse des intermuskulären Fettgewebes beim Menschen

Isolierung von Zellkernen aus humanem intermuskulärem Fettgewebe und nachgeschaltete Einzelkern-RNA-Sequenzierung

Our research group is trying to understand the cell composition and cell-specific transcriptional profiles of intermuscular adipose tissue or IMAT in humans. IMAT is actually an ectopic adipose depot residing between and around muscle fibers. Computed tomography and MRI have been able to quantify IMAT volume and have recently shown that it increases with both age and BMI, and is also associated with muscle degenerative diseases.In vitro secretome studies have recently shown that IMAT secreted proteins have the ability to influence insulin sensitivity, inflammatory profiles, and also contractile protein expressions in human skeletal muscle. The limited amount of IMAT tissue collected during biopsies and the lipid laden nature of adipocytes make this tissue incompatible with tissue digestion and single cell RNA-Seq. Therefore, we wanted to develop a protocol that performed nuclei isolation on small amounts of tissue that could then be used in downstream single nuclei RNA-Seq.This protocol will enable researchers to determine the compositional and cell-specific transcriptional alterations that occur in IMAT in different metabolic disease states and in response to interventions. We hope that this information will be crucial in developing new therapeutic strategies to help alleviate metabolic disease by either targeting IMAT directly or its secreted factors. The field severely lacks data on the biology of IMAT, its cellular origins, and communication with other cells, resident both within and outside of IMAT, as well as its molecular responses to lifestyle interventions, such as diet and exercise or pharmacotherapy.All of these are key research questions for our group.

Pulverisierung und Homogenisierung von gefrorenen humanen intermuskulären Fettgewebsproben für die Zellkernisolierung 

Füllen Sie zunächst den Kanister mit flüssigem Stickstoff, platzieren Sie Mörser, Stößel, Mikroschaufeln, Spatel, Glasböden, Edelstahlstößel und automatischen Downer auf der Arbeitsplattform. Die Gläser in einem mit Eis gefüllten Becherglas vorkühlen. Füllen Sie die beiden Mörser mit dem Stößel und dem Spatel mit flüssigem Stickstoff, um die Instrumente abzukühlen.In der Zwischenzeit einen Milliliter Homogenisierungspuffer in die Glasböden geben. Füllen Sie beide Mörser mit flüssigem Stickstoff und gießen Sie die 50 Milligramm intermuskulärer Fettgewebsprobe in einen der Mörser. Zum Pulverisieren drücken Sie den Stößel vorsichtig auf das Gewebestück, um es in kleine Stücke zu brechen.Sobald das Gewebe pulverisiert ist, bleibt noch ein Viertel bis die Hälfte eines Mörsers mit flüssigem Stickstoff übrig. Kippen Sie den Mörser, um pulverisiertes Gewebe in der Lippe des Mörsers aufzufangen. Sobald der flüssige Stickstoff verdampft ist, schöpfen Sie das pulverisierte Gewebe in die Glasböden mit dem Homogenisierungspuffer.Auf dem automatischen Downer. Bewegen Sie das Glas auf dem Edelstahlstößel 10 Mal in Vorwärtsrichtung auf und ab, gefolgt von 10 Hüben in umgekehrter Richtung. Sobald die Lösung trüb und frei von sichtbaren Gewebefragmenten ist, geben Sie sie in ein vorgekühltes 1,7-Milliliter-Röhrchen mit geringer Bindung auf Eis.Spülen Sie die Daunen mit 400 Mikrolitern Homogenisierungspuffer ab und geben Sie ihn in ein Röhrchen mit geringer Bindung.

pulverization-homogenization-frozen-human-intermuscular-adipose

Research

JoVE Journal

Biology

Pulverization and Homogenization of Frozen Human Intermuscular Adipose Tissue Samples for Nuclei Isolation

67 Aufrufe.  AdventHealth. Füllen Sie zunächst den Kanister mit flüssigem Stickstoff, platzieren Sie Mörser, Stößel, Mikroschaufeln, Spatel, Glasböden, Edelstahlstößel und automatischen Downer auf der Arbeitsplattform. Die Gläser in einem mit Eis gefüllten Becherglas vorkühlen. Füllen Sie die beiden Mörser mit dem Stößel und dem Spatel mit flüssigem Stickstoff, um die Instrumente abzukühlen.In der Zwischenzeit einen Milliliter Homogenisierungspuffer in die Glasböden geben. Füllen Sie beide M?...

Serie: Pulverisierung und Homogenisierung von gefrorenen humanen intermuskulären Fettgewebsproben für die Zellkernisolierung 

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose depot.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and ...