Name: Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure (Video) | JoVE Journal
Uploaded: 2019-08-23T13:30:00.000+00:00
Duration: 8 s
Description: Learn to isolate and characterize human muscle progenitor cells from skeletal muscle biopsy tissue. This method enables researchers to study proliferation, differentiation, and regenerative processes in human myogenesis.

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The authors thank the Cornell University, Biotechnology Resource Center Imaging Facility for their help with fluorescence activated cell sorting. We also thank Molly Gheller for her help with participant recruitment and Erica Bender for conducting the skeletal muscle biopsies. Finally, we thank the participants for their time and participation in the study.This work was supported by the National Institute on Aging of the National Institutes of Health under Award Number R01AG058630 (to B.D.C. and A.E.T.), by a Glenn Foundation for Medical Research and American Federation for Aging Research Grant for Junior Faculty (to B.D.C.), and by the President&#39;s Council for Cornell Women (to A.E.T.).

<ol><li>Janssen, I., Heymsfield, S. B., Wang, Z., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+mass+and+distribution+in+468+men+and+women+aged+18-88+yr%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr.</a> Journal of Applied Physiology. 89 (1), 81-88 (2000).</li>
<li>D'Souza, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+satellite+cell+number+and+function+in+humans+and+mice+with+type+1+diabetes+is+the+result+of+altered+notch+signaling%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Decreased satellite cell number and function in humans and mice with type 1 diabetes is the result of altered notch signaling.</a> Diabetes. 65 (10), 3053-3061 (2016).</li>
<li>Scheele, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Satellite+cells+derived+from+Obese+humans+with+type+2+diabetes+and+differentiated+into+Myocytes+in+vitro+exhibit+abnormal+response+to+IL-6%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Satellite cells derived from Obese humans with type 2 diabetes and differentiated into Myocytes in vitro exhibit abnormal response to IL-6.</a> PLoS One. 7 (6), e39657(2012).</li>
<li>Riddle, E. S., Bender, E. L., Thalacker-Mercer, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+capacity+of+human+muscle+progenitor+cells+differs+by+age%2C+sex%2C+and+metabolic+fuel+preference%5BTitle%5D)+AND+%22American+Journal+of+Physiology%3A+Cell+Physiology%22%5BJournal%5D)">Expansion capacity of human muscle progenitor cells differs by age, sex, and metabolic fuel preference.</a> American Journal of Physiology: Cell Physiology. 315 (5), C643-C652 (2018).</li>
<li>Charville, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+expansion+and+in+vivo+self-renewal+of+human+muscle+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Ex vivo expansion and in vivo self-renewal of human muscle stem cells.</a> Stem Cell Reports. 5 (4), 621-632 (2015).</li>
<li>Alexander, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD82+Is+a+Marker+for+Prospective+Isolation+of+Human+Muscle+Satellite+Cells+and+Is+Linked+to+Muscular+Dystrophies%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies.</a> Cell Stem Cell. 19 (6), 800-807 (2016).</li>
<li>Thalacker-Mercer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cluster+analysis+reveals+differential+transcript+profiles+associated+with+resistance+training-induced+human+skeletal+muscle+hypertrophy%5BTitle%5D)+AND+%22Physiological+Genomics%22%5BJournal%5D)">Cluster analysis reveals differential transcript profiles associated with resistance training-induced human skeletal muscle hypertrophy.</a> Physiological Genomics. 45 (12), 499-507 (2013).</li>
<li>Garcia, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Yield+Purification%2C+Preservation%2C+and+Serial+Transplantation+of+Human+Satellite+Cells%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells.</a> Stem Cell Reports. 10 (3), 1160-1174 (2018).</li>
<li>Yokoyama, W. M., Thompson, M. L., Ehrhardt, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreservation+and+thawing+of+cells%5BTitle%5D)+AND+%22Current+Protocols+in+Immunology%22%5BJournal%5D)">Cryopreservation and thawing of cells.</a> Current Protocols in Immunology. , Appendix 3, 3G (2012).</li>
<li>Bareja, A., Billin, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Satellite+cell+therapy+-+from+mice+to+men%5BTitle%5D)+AND+%22Skeletal+Muscle%22%5BJournal%5D)">Satellite cell therapy - from mice to men.</a> Skeletal Muscle. 3 (1), 2(2013).</li>
<li>Riddle, E. S., Bender, E. L., Thalacker-Mercer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcript+profile+distinguishes+variability+in+human+myogenic+progenitor+cell+expansion+capacity%5BTitle%5D)+AND+%22Physiological+Genomics%22%5BJournal%5D)">Transcript profile distinguishes variability in human myogenic progenitor cell expansion capacity.</a> Physiological Genomics. 50 (10), 817-827 (2018).</li>
<li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Satellite+Cell+Transplantation+and+Regeneration+from+Diverse+Skeletal+Muscles%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles.</a> Stem Cell Reports. 5 (3), 419-434 (2015).</li>
</ol>

The authors have nothing to disclose.

Primary hMPCs are an important research model used to understand skeletal muscle biology and the regenerative process. Additionally, hMPCs have the potential to be used for therapy. However, there are recognized challenges in using primary hMPCs for both research and therapy, including the limited understanding of cells derived from humans10. There is also a large degree of variation in the expansion capacity among donor cultures, which limits the potential for use of hMPCs and can affect research...

Skeletal muscle is the largest organ system in the human body, accounting for 30&#8722;40% of whole body mass1. In addition to its well-recognized role in locomotion, skeletal muscle maintains body temperature and posture, and plays a central role in whole body nutrient homeostasis. Research involving human participants, animals, and cell culture models are all valuable to address questions pertaining to skeletal muscle biology and regeneration. Isolation and culture of human primary muscle progenitor cells (hMPCs) provides a robust model that allows for cell culture techniques and manipulations to be applied to human samples. An advantage of using hMPCs is that they retain the genetic and metabolic phenotype from each donor2,3. Maintenance of the donor phenotype allows researchers to examine inter-individual variation in the myogenic process. For example, we have employed our hMPC characterization method to identify age- and sex-related differences in hMPC population expansion capacity4.
The purpose of this protocol is to detail techniques to isolate, culture, characterize, and differentiate&#160;hMPCs from skeletal muscle biopsy tissue. Building on previous work that described hMPCs and identified potential cell surface makers for hMPC isolation5,6, this protocol fills a critical gap in knowledge by linking the isolation to the characterization of hMPCs. Further, the detailed step-by-step instructions included in this protocol make hMPC isolation and characterization accessible to a broad scientific audience, including those with limited prior experience with hMPCs. Our protocol is among the first to describe use of an imaging cytometer to track cell populations. Newly designed imaging cytometers are state-of-the-art, high-throughput, and microplate-based, enabling live cell imaging, cell counting, and multichannel fluorescence analysis of all cells in each well of a culture vessel within minutes. This system allows for rapid quantification of dynamic changes in proliferation and viability of an entire cell population with only minimal disruption to the culture. For example, we are able to perform objective measures of confluence on successive days in vitro to determine growth kinetics of each culture derived from different donors. Many protocols in the literature, particularly those involving the differentiation of MPCs, require cells to reach a defined level of confluence before initiating differentiation or treatment7. Our method allows for objective determination of the confluence of each well in a culture vessel allowing researchers to initiate treatment in an unbiased, non-subjective manner.
In the past, a major limitation of using primary hMPCs was low yields that limit the number of cells available for experiments. We and others have shown the yield of MPCs from skeletal muscle biopsy tissue is 1&#8722;15 MPCs per milligram of tissue (Figure 1)8. Because our protocol allows for four passages of the cells prior to purification with fluorescence activated cell sorting (FACS), our cryopreserved hMPC yields, derived from small amounts of biopsy tissue (50&#8722;100 mg), are sufficient to address research aims where multiple experiments are required. Our FACS protocol produces a ~80% pure (Pax7 positive) MPC population, thus our protocol is optimized for both yield and purity.

<table><tbody><tr><td>0.25% Trypsin, 2.21 mM EDTA</td><td>Corning</td><td>25-053-Cl</td><td>Trypsin used for removing adherent &lt;em&gt;h&lt;/em&gt;MPCs from cell culture vessels</td></tr><tr><td>10 cm cell culture plate</td><td>VWR</td><td>664160</td><td>Plates used for culturing &lt;em&gt;h&lt;/em&gt;MPCs</td></tr><tr><td>15 mL Falcon tube</td><td>Falcon</td><td>352196</td><td>15 mL conical tubes used throughout the &lt;em&gt;h&lt;/em&gt;MPC isolation and culturing protocols</td></tr><tr><td>24 well cell culture plate</td><td>Grenier Bio-One</td><td>662 160</td><td>Plates used for culturing &lt;em&gt;h&lt;/em&gt;MPCs</td></tr><tr><td>7-AAD Viability Staining Solution</td><td>eBioscience</td><td>00-6993-50</td><td>Viability stain for identifying living cells during FACS sorting</td></tr><tr><td>Alexa Fluor 488 anti-human CD29, Clone: TS2/16</td><td>BioLegend</td><td>303016</td><td>Conjugated antibody for FACS&amp;nbsp;</td></tr><tr><td>Black 96-well cell culture plate</td><td>Grenier Bio-One</td><td>655079</td><td>96-well cell culture plate ideal for fluorescent imaging using the Celigo S</td></tr><tr><td>Celigo S</td><td>Nexcelcom Bioscience</td><td></td><td>Imaging cytometer used to track &lt;em&gt;h&lt;/em&gt;MPC cultures</td></tr><tr><td>Cell Strainer</td><td>VWR</td><td>352350</td><td>Cell strainer to eliminate large pieces of debris during muscle biopsy processing</td></tr><tr><td>Collagen Type I (Rat Tail)</td><td>Corning</td><td>354236</td><td>Collagen for coating cell culture plates&amp;nbsp;</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11 088 882 001</td><td>Used for degradation of collagen and other connective tissue in the skeletal muscle biopsy tissue</td></tr><tr><td>Dimethyl Sulfoxide</td><td>VWR</td><td>WN182</td><td>Used for cryopreservation of &lt;em&gt;h&lt;/em&gt;MPCs</td></tr><tr><td>Dispase II</td><td>Sigma Life Sciences</td><td>D4693</td><td>A protease used for enzymatic digestion of skeletal muscle biopsy tissue</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium Low Glucose powder</td><td>Gibco</td><td>31600-034</td><td>Low glucose DMEM for muscle biopsy processing</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Gibco</td><td>21600-010</td><td>PBS for muscle biopsy processing</td></tr><tr><td>EDTA Disodium Salt Dihydrate</td><td>J.T. Baker</td><td>4040-01&amp;nbsp;</td><td>Required for FACS buffer</td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>89510-186&amp;nbsp;</td><td>Fetal bovine serum used for &lt;em&gt;h&lt;/em&gt;MPC growth media</td></tr><tr><td>Ham&#039;s F12</td><td>Gibco</td><td>21700-026</td><td>Base media for &lt;em&gt;h&lt;/em&gt;MPCs</td></tr><tr><td>Heat Inactivated Equine Serum</td><td>Gibco</td><td>26-050-070</td><td>Horse serum used to make &lt;em&gt;h&lt;/em&gt;MPC differentiation media</td></tr><tr><td>Hemocytometer</td><td>iNCyto</td><td>DHC-N0105</td><td>Used to count cells</td></tr><tr><td>Hibernate A</td><td>Gibco</td><td>A1247501</td><td>Media for preserving skeletal muscle biopsy tissue</td></tr><tr><td>Hoechst 33342, trihydrochloride, trihydrate</td><td>Life Technologies</td><td>H21492</td><td>DNA stain for identifying all cells using the Celigo S</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A416P-4</td><td>Used for controlled rate freezing of &lt;em&gt;h&lt;/em&gt;MPCs</td></tr><tr><td>Moxi buffer</td><td>Orflo</td><td>MXA006</td><td>Buffer for automated cell counter</td></tr><tr><td>Moxi Cassettes</td><td>Orflo</td><td>MXC002</td><td>Cassesttes for automated cell counter</td></tr><tr><td>Moxi z Mini Automated Cell Counter</td><td>Orflo</td><td></td><td>Automated cell counter</td></tr><tr><td>Mr. Frosty Freezing Container</td><td>Thermo Fisher Scientific</td><td>5100-0001</td><td>Commerically available controlled rate cell freezing container</td></tr><tr><td>Normal Goat Serum (10%)</td><td>Thermo Fisher Scientific</td><td>50062Z</td><td>Goat serum used in FACS buffer</td></tr><tr><td>PE-Cy7 Mouse Anti-human CD56 , Clone: B159</td><td>BD Pharmingen</td><td>557747</td><td>Conjugated antibody for FACS&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin 100x Solution</td><td>Corning</td><td>30-002-CI</td><td>Antibiotics added to culture media</td></tr><tr><td>Propidium iodide</td><td>Thermo Fisher Scientific</td><td>P3566</td><td>DNA stain for identifying dead cells using the Celigo S</td></tr><tr><td>Recombinant Human basic fibroblast growth factor</td><td>Promega</td><td>G5071</td><td>Supplement in &lt;em&gt;h&lt;/em&gt;MPC growth media to prevent spontaneous differentiation</td></tr><tr><td>Recovery Cell Culture Freezing Medium&amp;nbsp;</td><td>Gibco</td><td>12648-010</td><td>Media used to cryoperseve muscle biopsy slurries</td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher Scientific</td><td>S233-3</td><td>Added to Ham&#039;s F12</td></tr><tr><td>Sterile Round Bottom 5 mL tubes</td><td>VWR</td><td>60818-565</td><td>Tubes used for FACS</td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td>Compensation beads fort calibrating flow FACS settings</td></tr></tbody></table>

isolation, culture, characterization, and differentiation of human muscle progenitor cells from the skeletal muscle biopsy procedure

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

Representative flow cytometry results of hMPC isolation from human muscle tissue can be viewed in Figure 1. hMPCs can be identified by first gating events based on side scatter and forward scatter to eliminate dead cells or debris, followed by selecting only cells which are negative for 7-AAD and therefore are viable. Selection of cells positive for both the cell surface markers CD56 and CD29 represents the hMPC population. A biopsy of 60 mg only provides app...

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

isolation-culture-characterization-differentiation-human-muscle

Research

JoVE Journal

Developmental Biology

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purification of Progenitors from Skeletal Muscle

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation.  Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue.  A technique for the immortalization of these cell lines is also described.  Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2´-deoxyuridine incorporation assay.

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

This protocol provides a standardized method for isolating and enriching primary myoblasts from adult and neonatal mouse skeletal muscle through tissue dissociation, sequential filtration, preplating, and defined culture conditions, enabling the establishment and maintenance of satellite cell-derived myoblast cultures for downstream experimental applications.

Isolation of Skeletal Muscle Satellite Cells for In Vitro Myogenesis Studies

Learn to isolate and characterize human muscle progenitor cells from skeletal muscle biopsy tissue. This method enables researchers to study proliferation, differentiation, and regenerative processes in human myogenesis.

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure (Video) | JoVE Journal

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

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