Isolating adult stem cells from musculoskeletal soft tissues based on the cell’s adherence speed to flask.
In general, the complete modified preplate procedure requires 1-2 days of preparation, 1 week of performing the technical procedure, 2-3 days of cell identification with immunocytochemistry, and an additional 2-3 weeks of stem cell population expansion. The equipment required for stem cell culture are similar to that of other cell culture systems which includes a bench-top centrifuge, CO2 incubator, Laminar flow tissue culture hood and an inverted microscope equipped with fluorescence and a digital camera. The isolated stem cells also can be cloned and can be stored long-term in liquid nitrogen for the current or future studies 1,2. All reagents and materials used for this procedure must be sterile and handled aseptically.
Step 1: Preparation
Step 2: Tissue biopsies, isolation and mechanical dissociation1-4
The tissue biopsies are performed under a sterile culture hood and include freshly harvested tendons and skeletal muscles are obtained from the tibia or soleus muscles of the hindlimb of wild-type mice (Female C57BL/6J, 4-5 weeks of age or younger, Jackson Laboratory). Any visible connective tissue, blood vessels, and adipose tissue are then removed from the biopsy samples. The tissues of tendon and muscle are then carefully identified under a surgical dissecting microscope to remove remnants of skin and bone and placed in a dish containing cold (4°C) HBSS (supplemented with add 5 % of FBS). The isolated tissues are then separately placed on collagen coated dishes containing cold HBSS and will be minced (<1×1 mm3) into a coarse slurry using micro-scissors and/or scalpel blades.
Step 3: Enzymatic digestion of the tissues:
The minced tissue is then enzymatically dissociated through a series of steps2,4,5
Step 4: Preplating to isolate different cell populations based on cell adhesion rate1-3
Step 5: Identification and expansion of isolated stem cells
It has been recognized that some adult tissues contain multiple stem cells. Over the past few years of study, stem cells have been detected in the soft musculoskeletal tissues, including skeletal muscle and tendon. This current protocol details a procedure, known as the modified preplate technique, that has been successfully used in our laboratory to isolate adult stem cells from tendons and skeletal muscle. Using the modified preplate technique described above the cells can be divided into several populations based on their adhesion characteristics to collagen coated flasks (Figure 1). Fibroblasts and myofibroblasts found within the tissues quickly adhere to the collagen coated flasks within 24-48 hours and are initially separated from other cells in PP1-PP2. The myogenic or endothelial cells adhere to collagen coated flasks after a longer time period, within 48-96 hours (PP3-PP5), and can be harvested and identified by their cell surface markers. The later preplated cells, 96 hours or later (PP6), are regarded as adult stem cells (Figure 2). The late preplate cells appear small, round, and translucent at the beginning of their isolation and can be further characterized by flow cytometry or immunocytochemistry for their expression of stem cell antigen 1 (Sca1), CD34, fetal liver kinase 1 (Flk1), and other stem cell markers (Figure 3). The isolated stem cells have the capacity for self-renewal and experimental studies have demonstrated their multipotency through their differentiation into the three germ layers: mesoderm, ectoderm, and endoderm 9. Multiple investigations have also revealed that these stem cells differentiate into cells of the mesoderm lineage including osteocytes, adipocytes, chondrocytes, and hematopoietic cells 6,8. Other studies have demonstrated that the adult stem cells differentiate into ectoderm cell lineages through their expression of neuronal and glial cell markers and their ability to improve limb function after the peripheral nerve has been damaged 10. These isolated adult stem cells have been beneficial for repairing injured and diseased musculoskeletal tissues, and other related in vivo studies have been performed on other tissues including heart, liver, and spinal cord as well as specific medical conditions like small intestinal submucosa and bladder to treat stress urinary incontinence 9.
Figure 1.
Figure 2.
Figure 3.
The authors have nothing to disclose.
The authors are greatly appreciative of Ms. Haiying Pan and Mr. Ian Bellayr for their technical help on cell isolation for the current films and Mr. Kyle Holden and Mr. Jonathan Lucas for their help on slide editing. Many thanks to the following list of persons who have been involved with the development of the modified preplate techniques over the past years: Drs. Burhan Gharaibeh, BaoHong Cao, Deasy Bridget, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, Kimimasa Tobita, Ron Jankowski, Makato Ikezawa. We are grateful for the technical assistance from James H. Cummins, Ryan Pruchnic, Joseph Feduska, Rebecca C. Schugar, Haiying Pan and Jessica Tebbets. The author also would like to acknowledge the financial support for this work from the Muscular Dystrophy Association (MDA), the National Institutes of Health (NIH), the Department of Defense (DOD), the Children’s Hospital of Pittsburgh of UPMC, the Department of Orthopaedic Surgery, and the University of Pittsburgh.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM | Invitrogen | #11995-073 | ||
FBS | Invitrogen | #10437-028 | ||
Heat-inactivated horse serum | Invitrogen | #26050-088 | ||
Chick embryo extract | Accurate Chemical | #CE650T-10 | ||
100U ml penicillin/streptomycin | Invitrogen | #15140-122 | ||
Dulbecco’s PBS | Invitrogen | #14190-250 | ||
Hank’s Buffered Salt Solution | Invitrogen | #24020-117 | ||
Collagenase type XI 0.2%(wt/vol) | Sigma-Aldrich | #C7657 | ||
Dispase2.4U ml | Invitrogen | #17105-041 | ||
Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250) | Invitrogen | #15400-054 | ||
Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter | Sigma-Aldrich | #C-9791 | ||
DMSO | Sigma | #D-2650 |