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

유전자 발현 프로파일 연구를 위한 IMAT에서 단일 세포 핵 분리 

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.

저자 스포트라이트: 인간의 근육 간 지방 조직의 세포 미스터리 해독

인간 근육 간 지방 조직에서 핵 분리 및 다운스트림 단일 핵 RNA 염기서열분석

핵 분리를 위한 Frozen Human Intermuscular Adipose Tissue Samples의 분쇄 및 균질화 

시작하려면 작업 플랫폼에 액체 질소 플레이스 모르타르, 유봉 마이크로 스쿱, 주걱, 유리 다운, 스테인리스 스틸 유봉 및 자동 다우너를 채웁니다. 얼음을 채운 비커에서 유리 다운을 미리 식힙니다. 유봉과 주걱이 들어있는 두 개의 모르타르에 액체 질소를 채워 기구를 식힙니다.한편, 1mL의 균질화 완충액을 글라스 다운에 추가합니다. 두 모르타르에 액체 질소를 채우고 50mg의 인간 근육 간 지방 조직 샘플을 모르타르 중 하나에 붓습니다. 분쇄를 위해 유봉을 티슈 조각에 부드럽게 눌러 작은 조각으로 나눕니다.조직이 분쇄되면 액체 질소 모르타르의 1/4에서 절반이 남습니다. 모르타르의 입술에 분쇄된 조직을 모으는 모르타르를 기울입니다. 액체 질소가 증발하면 분쇄된 조직을 균질화 완충액이 들어 있는 유리 다운에 퍼넣습니다.자동 다우너에. 유리를 스테인리스 스틸 유봉을 위아래로 움직여 정방향으로 10번 친 다음 반대 방향으로 10번 움직입니다. 용액이 뿌옇고 눈에 보이는 조직 조각이 없어지면 얼음 위에서 미리 냉각된 1.7밀리리터의 낮은 바인드 튜브로 옮깁니다.400 마이크로리터의 균질화 완충액으로 다운을 헹구고 로우 바인드 튜브로 옮깁니다.

pulverization-homogenization-frozen-human-intermuscular-adipose

Research

JoVE Journal

Biology

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

68 조회수. 시작하려면 작업 플랫폼에 액체 질소 플레이스 모르타르, 유봉 마이크로 스쿱, 주걱, 유리 다운, 스테인리스 스틸 유봉 및 자동 다우너를 채웁니다. 얼음을 채운 비커에서 유리 다운을 미리 식힙니다. 유봉과 주걱이 들어있는 두 개의 모르타르에 액체 질소를 채워 기구를 식힙니다.한편, 1mL의 균질화 완충액을 글라스 다운에 추가합니다. 두 모르타르에 액체 질소를 채우?...

비디오: 핵 분리를 위한 Frozen Human Intermuscular Adipose Tissue Samples의 분쇄 및 균질화 

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 ...