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 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 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.
Skeletal muscle is the largest organ system in the human body, accounting for 30−40% 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 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 1−15 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 (50−100 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.
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
2. Isolating Human Muscle Progenitor Cells from Biopsy Tissue
3. Culturing Human Muscle Progenitor Cells
4. Isolating Pax7+ Human Muscle Progenitor Cells via Flow Cytometry
5. Characterizing Human Muscle Progenitor Cell (hMPC) Cultures Using an Imaging Cytometer
6. Differentiating Human Muscle Progenitor Cells (hMPCs) to Myotubes
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 approximately 75−250 hMPCs.
A representative confluence scan is shown in Figure 2A. The green outlines in Figure 2B were generated by the imaging cytometer and show how the imaging cytometer determined confluence based on the analysis settings selected. In both images shown, the confluence determined by the imaging cytometer agrees with the confluence visually determined by the user. However, these images also highlight that the imaging cytometer is not perfect. For example, the red arrow highlights an imperfection in the plate which is being counted as cells. If a large number of these imperfection are present on the plate, the resulting confluence will not accurately represent the confluence of the cells. Figure 2C shows a representation of all three channels used to count cells (brightfield, blue and red) and the merged image. Figure 2D shows heterogeneity between donors. Discovering and accurately characterizing this heterogeneity is a key application of the imaging cytometer. Figure 2E shows that confluence measurements and nuclei counts from unique donors are highly correlated.
Figure 3 provides a guideline for how to identify hMPCs based on the FACS procedure described in this protocol. Gating based on forward and side scatter allows for separation of viable cells from debris (Figure 3A). A comparison of forward scatter by height to forward scatter by area was used to distinguish events representing single cells (the boxed area in Figure 3B). Viable cells are marked by a lack of incorporation of the viability stain 7-AAD (Figure 3C). Finally, cells positive for both CD56 and CD29 are identified as hMPCs (Figure 3D). To validate the FACS procedure described in this protocol the selection of Pax7 positive cells can be determined by immunostaining cells with a Pax7-specific antibody and measuring expression on a flow cytometer. Figure 4 compares the number of hMPCs as determine by Pax7 immunostaining (Figure 4A) vs. the FACS procedure (Figure 4B) detailed in this protocol from the same population of cells. Figure 5 uses Pax7 immunostaining and analysis via flow cytometry to show the enrichment of Pax7 expressing cells in the total population after FACS (Figure 5A compared to Figure 5B) as well as the maintenance of the number of Pax7 expressing cells after passaging (Figure 5B compared to Figure 5C).
hMPCs can be differentiated to form myotubes by following section 6. To determine whether isolated hMPCs maintain myogenic capacity cells can be immunostained with an antibody specific for embryonic myosin heavy chain and visualized using fluorescent microscopy. Representative images of embryonic myosin heavy chain positive myotubes can be viewed in Figure 6.
Figure 1: Representative flow cytometry images for hMPCs sorted directly out of muscle biopsy tissue. (A) Side scatter area (y-axis) vs. forward scatter area (x-axis) gating strategy used to identify all cells in a donor sample. (B) 7-AAD viability stain (y-axis) vs. forward scatter (x-axis) to identify live cells. (C) Negative selection sorting marker profile (-CD11b, -CD31, -CD45, -GlyA [y-axis]) vs. selection marker CD34. (D) Negative selection marker CD34 (y-axis) vs. the positive selection marker CD29. (E)Positive selection marker CD56 (y-axis) vs. the positive selection marker CD29 (x-axis). (F)Yields from three different skeletal muscle biopsies. FSC = forward scatter; SSC = side scatter; hMPCs, human muscle progenitor cells.
Figure 2: Proliferative potential of hMPCs derived from human donors is maintained after 6 passages. (A)Representative confluence scan. (B) Representative confluence scan with green outlines showing how the imaging cytometer determined confluence. The red arrow shows an imperfection on the plate. (C) Representative brightfield, Hoechst 33342 (blue), and propidium Iodide (red) staining and a merged image of these three channels. Scale bars = 1mm. (D) Nuclei counts from 6 unique donors highlights hMPC heterogeneity. (E) Confluence and nuclei count are highly correlated. Please click here to view a larger version of this figure.
Figure 3: Representative flow cytometry sorting parameters for hMPC isolation. (A) Side scatter area (y-axis) vs. forward scatter area (x-axis) gating strategy used to identify all cells in a donor sample. (B) Forward scatter height (y-axis) vs. forward scatter area (x-axis) to disqualify doublets from the sorting population. (C) Forward scatter height (y-axis) vs. 7-AAD incorporation to identify viable cells. (D) PE-Cy7 (CD56) vs. AF488 (CD29) staining with Q2 representing double positive cells (hMPCs). Color represents the density of events.
Figure 4: Comparison of CD29/CD56 positivity vs. Pax7 positivity in passage 4 hMPCs from the same donor. (A) Count (y-axis) vs. Pax7 expression (x-axis). (B)Positive selection marker CD56 (y-axis) vs. positive selection marker CD29 (x-axis). NCAM = neural cell adhesion molecule; hMPC = human muscle progenitor cell.
Figure 5: Pax7 positivity is maintained in hMPCs derived from the same donor over multiple passages. (A) Pax7 expression (x-axis) of cells which had been passaged 4 times prior to FACS sorting. (B) Pax7 expression (x-axis) of hMPCs 1 passage after FACS sorting for CD29 and CD56 positivity (5 total passages). (C) Pax7 expression (x-axis) of hMPCs 2 passages after FACS sorting for CD29 and CD56 positivity (6 total passages).
Figure 6: Staining of hMPC derived myotubes for embryonic myosin heavy chain. Representative microscopic images of differentiated hMPCs co-stained with DNA stain (Hoechst 33342, blue) and embryonic myosin heavy chain (green) (n = 3). Please click here to view a larger version of this figure.
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 results11. For example, it has recently been demonstrated that hMPCs isolated using the method described in this protocol produced cells that varied in expansion capacity and this was driven by transcriptional profile that did not align with sex or age11.
Here, we present an efficient and high yield method for isolating hMPCs in sufficient quantities for a number of downstream applications including differentiation into myotubes, thereby modeling the myogenic process in vitro. Our method relies on FACS to isolate CD56+/CD29+ cells, which have previously been identified as representing an enriched Pax7 expressing population12. Other equally valid cell sorting strategies have also been described5,6.
The most important aspect of our hMPC isolation and culture method is the careful monitoring of individual cultures. The donor-based heterogeneity includes number of hMPCs obtained from each biopsy, their time to initiate proliferation in vitro, and their population expansion rate. Therefore, if differences in myotube formation after a treatment is the question of interest individual, hMPC cultures should be switched to treatment based on a confluence level determined a priori and assessed objectively using the imaging cytometer.
Our method to isolate and culture hMPCs has been optimized to obtain yields of cells in the millions for in vitro experiments from relatively little starting biopsy tissue (50−100 mg). The major benefit of this approach is that multiple assays and experiments can be performed on hMPCs from the same donor, allowing for maintenance of donor phenotype across experiments while introducing little inter-experiment variability. Our method differs from recently published methods that focus on extracting the purest population of Pax7 expressing cells directly out of biopsy tissue where the focus is xenotransplantation8. The challenge with these previous methods however, is that they require a starting tissue weight of greater than one gram. Therefore, they are not practical for research involving human skeletal muscle biopsies. One limitation of our method is that the potential for xenotransplantation has not been assessed. However, this type of procedure was not one of the original intended downstream applications. Future directions of this method may include assessing the engraftment capacity of hMPCs, after undergoing our isolation procedure, to determine whether there is potential for use of this procedure in MPC transplantation studies.
The authors have nothing to disclose.
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's Council for Cornell Women (to A.E.T.).
0.25% Trypsin, 2.21 mM EDTA | Corning | 25-053-Cl | Trypsin used for removing adherent hMPCs from cell culture vessels |
10 cm cell culture plate | VWR | 664160 | Plates used for culturing hMPCs |
15 mL Falcon tube | Falcon | 352196 | 15 mL conical tubes used throughout the hMPC isolation and culturing protocols |
24 well cell culture plate | Grenier Bio-One | 662 160 | Plates used for culturing hMPCs |
7-AAD Viability Staining Solution | eBioscience | 00-6993-50 | Viability stain for identifying living cells during FACS sorting |
Alexa Fluor 488 anti-human CD29, Clone: TS2/16 | BioLegend | 303016 | Conjugated antibody for FACS |
Black 96-well cell culture plate | Grenier Bio-One | 655079 | 96-well cell culture plate ideal for fluorescent imaging using the Celigo S |
Celigo S | Nexcelcom Bioscience | Imaging cytometer used to track hMPC cultures | |
Cell Strainer | VWR | 352350 | Cell strainer to eliminate large pieces of debris during muscle biopsy processing |
Collagen Type I (Rat Tail) | Corning | 354236 | Collagen for coating cell culture plates |
Collagenase D | Roche | 11 088 882 001 | Used for degradation of collagen and other connective tissue in the skeletal muscle biopsy tissue |
Dimethyl Sulfoxide | VWR | WN182 | Used for cryopreservation of hMPCs |
Dispase II | Sigma Life Sciences | D4693 | A protease used for enzymatic digestion of skeletal muscle biopsy tissue |
Dulbecco's Modified Eagle Medium Low Glucose powder | Gibco | 31600-034 | Low glucose DMEM for muscle biopsy processing |
Dulbecco's Phosphate Buffered Saline | Gibco | 21600-010 | PBS for muscle biopsy processing |
EDTA Disodium Salt Dihydrate | J.T. Baker | 4040-01 | Required for FACS buffer |
Fetal Bovine Serum | VWR | 89510-186 | Fetal bovine serum used for hMPC growth media |
Ham's F12 | Gibco | 21700-026 | Base media for hMPCs |
Heat Inactivated Equine Serum | Gibco | 26-050-070 | Horse serum used to make hMPC differentiation media |
Hemocytometer | iNCyto | DHC-N0105 | Used to count cells |
Hibernate A | Gibco | A1247501 | Media for preserving skeletal muscle biopsy tissue |
Hoechst 33342, trihydrochloride, trihydrate | Life Technologies | H21492 | DNA stain for identifying all cells using the Celigo S |
Isopropanol | Fisher Scientific | A416P-4 | Used for controlled rate freezing of hMPCs |
Moxi buffer | Orflo | MXA006 | Buffer for automated cell counter |
Moxi Cassettes | Orflo | MXC002 | Cassesttes for automated cell counter |
Moxi z Mini Automated Cell Counter | Orflo | Automated cell counter | |
Mr. Frosty Freezing Container | Thermo Fisher Scientific | 5100-0001 | Commerically available controlled rate cell freezing container |
Normal Goat Serum (10%) | Thermo Fisher Scientific | 50062Z | Goat serum used in FACS buffer |
PE-Cy7 Mouse Anti-human CD56 , Clone: B159 | BD Pharmingen | 557747 | Conjugated antibody for FACS |
Penicillin/Streptomycin 100X Solution | Corning | 30-002-CI | Antibiotics added to culture media |
Propidium iodide | Thermo Fisher Scientific | P3566 | DNA stain for identifying dead cells using the Celigo S |
Recombinant Human basic fibroblast growth factor | Promega | G5071 | Supplement in hMPC growth media to prevent spontaneous differentiation |
Recovery Cell Culture Freezing Medium | Gibco | 12648-010 | Media used to cryoperseve muscle biopsy slurries |
Sodium Bicarbonate | Fisher Scientific | S233-3 | Added to Ham's F12 |
Sterile Round Bottom 5 mL tubes | VWR | 60818-565 | Tubes used for FACS |
UltraComp eBeads | eBioscience | 01-2222-42 | Compensation beads fort calibrating flow FACS settings |