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

Summary

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.

Abstract

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.

Introduction

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 u....

Protocol

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

1. Identify the vastus lateralis muscle via palpation, anatomical landmarks, and active contraction of the muscle.
2. Palpate the vastus lateralis by having the participant tighten their quadriceps muscle and find the belly of the muscle, about 1/3 of the way from the patella to the hip, and just ventrolateral to the femur.
3. Use a paper measuring tape to mark an area with a surgica....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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