Name: isolation, culture, characterization, and differentiation of human muscle progenitor cells from the skeletal muscle biopsy procedure
Uploaded: 2019-08-23T13:30:00
Description: Watch this Scientific Journal Video about Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure at JoVE.com

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.).

This protocol was approved by the Institutional Review Board at Cornell University. All participants were screened for underlying health conditions and gave informed consent.
1. Obtaining Human Muscle Tissue via the Skeletal Muscle Biopsy
<ol>
	<li>Identify the vastus lateralis muscle via palpation, anatomical landmarks, and active contraction of the muscle.</li>
	<li>Palpate the vastus lateralis by having the participant tighten their quadriceps muscle and find the belly of the muscle, abou...

<ol>
	<li>Janssen, I., Heymsfield, S. B., Wang, Z., Ross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+mass+and+distribution+in+468+men+and+women+aged+18-88+yr.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+satellite+cell+number+and+function+in+humans+and+mice+with+type+1+diabetes+is+the+result+of+altered+notch+signaling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+derived+from+Obese+humans+with+type+2+diabetes+and+differentiated+into+Myocytes+in+vitro+exhibit+abnormal+response+to+IL-6.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+capacity+of+human+muscle+progenitor+cells+differs+by+age,+sex,+and+metabolic+fuel+preference.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+and+in+vivo+self-renewal+of+human+muscle+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD82+Is+a+Marker+for+Prospective+Isolation+of+Human+Muscle+Satellite+Cells+and+Is+Linked+to+Muscular+Dystrophies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cluster+analysis+reveals+differential+transcript+profiles+associated+with+resistance+training-induced+human+skeletal+muscle+hypertrophy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Yield+Purification,+Preservation,+and+Serial+Transplantation+of+Human+Satellite+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+thawing+of+cells.">Cryopreservation and thawing of cells.</a> Current Protocols in Immunology. , (2012).</li><li>Bareja, A., Billin, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+therapy+-+from+mice+to+men.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+profile+distinguishes+variability+in+human+myogenic+progenitor+cell+expansion+capacity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Satellite+Cell+Transplantation+and+Regeneration+from+Diverse+Skeletal+Muscles.">Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles.</a> Stem Cell Reports. 5 (3), 419-434 (2015).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 hMPCs 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 hMPCs</td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352196</td>
			<td>15 mL conical tubes used throughout the hMPC 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 hMPCs</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&#160;</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 hMPC 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&#160;</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 hMPCs</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&#39;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&#39;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&#160;</td>
			<td>Required for FACS buffer</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>89510-186&#160;</td>
			<td>Fetal bovine serum used for hMPC growth media</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Gibco</td>
			<td>21700-026</td>
			<td>Base media for hMPCs</td>
		</tr>
		<tr>
			<td>Heat Inactivated Equine Serum</td>
			<td>Gibco</td>
			<td>26-050-070</td>
			<td>Horse serum used to make hMPC 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 hMPCs</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&#160;</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 hMPC growth media to prevent spontaneous differentiation</td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium&#160;</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&#39;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

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...

Watch this Scientific Journal Video about Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure at JoVE.com

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

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 ...

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