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

从 IMAT 分离单细胞核用于研究基因表达谱 

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.

作者聚焦：破译人类肌间脂肪组织的细胞奥秘

从人肌间脂肪组织中分离细胞核和下游单核 RNA 测序

用于细胞核分离的冷冻人肌间脂肪组织样品的粉碎和均质化 

首先，用液氮填充罐，将研钵、研杵、微勺、刮刀、玻璃杵、不锈钢杵和自动向下料放在工作平台上。在装满冰块的烧杯中预冷玻璃杯。用液氮填充装有杵和刮刀的两个研钵，以冷却仪器。同时，向玻璃杯中加入一毫升均质缓冲液。用液氮填充两个研钵，并将 50 毫克人肌间脂肪组织样品倒入其中一个研钵中。粉碎时，将研杵轻轻按压在组织片上，将其掰成小块。一旦组织被粉碎，还剩下四分之一到一半的液氮研钵。倾斜研钵，将粉碎的组织收集在研钵的边缘。一旦液氮蒸发，将粉碎的组织舀入含有均质缓冲液的玻璃杯中。在自动下降器上。将玻璃杯在不锈钢杵上上下移动 10 次，然后向反向移动 10 次。一旦溶液浑浊且没有任何可见的组织碎片，将其转移到预冷的 1.7 毫升低结合管中，放在冰上。用 400 微升均质缓冲液冲洗羽绒服，并将其转移到低结合管中。

pulverization-homogenization-frozen-human-intermuscular-adipose

Research

JoVE Journal

Biology

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

68 次观看. 首先，用液氮填充罐，将研钵、研杵、微勺、刮刀、玻璃杵、不锈钢杵和自动向下料放在工作平台上。在装满冰块的烧杯中预冷玻璃杯。用液氮填充装有杵和刮刀的两个研钵，以冷却仪器。同时，向玻璃杯中加入一毫升均质缓冲液。用液氮填充两个研钵，并将 50 毫克人肌间脂肪组织样品倒入其中一个研钵中。粉碎时，将研杵轻轻按压在组织片上，将其掰成小块。?...

视频: 用于细胞核分离的冷冻人肌间脂肪组织样品的粉碎和均质化 

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 ...