The overall goal of this procedure is to isolate enriched populations of skeletal muscle stem cells from micro dissected tissue implants obtained from adult muscle or embryos in order to study their behavior and function. These cells may also have a therapeutic use. This is accomplished by first dissecting out target muscles from adult mouse carcasses or embryos from uterine tissue under aseptic conditions.
The second step of the procedure is to wash and clean muscle tissues for adult muscle tissues, dissect out fat and connective tissues and transfer into fresh DF 20 medium for embryos. Remove outer membranes and internal organs and transfer into fresh DF 20. Medium, then dissect to isolate muscle rich regions.
The third step of the procedure is to microdisect isolated tissues into 400 micrometer cubes. Transfer explan singly into the center 60 wells of a 96 well plate and incubate at 37 degrees Celsius. The final step of the procedure is to score the cells.
Cells will begin to emerge from successful x explants between a few hours or up to two weeks, postex explan depending on tissue origin. Ultimately, results can be obtained that show cells beginning to emerge from successful X explan cells can be expanded and subsequently used for a range of different in vitro and in vivo purposes, including proliferation and apoptosis, studies and identification of protein markers. These cells can also be clonally derived, genetically modified, and ultimately transplanted into a host for the study of regeneration or to test therapeutic applications.
Hi, I'm Janet Smith from my laboratory of skeletal muscle stem cell biology School of Biosciences, university of Birmingham. I'm Dean Lana from Janet Smith's lab And I'm Byron Chen, also from Janet Smith's lab. Today we'll show you a procedure for obtaining skeletal muscle stem cell cultures from Microdisect X fats obtained from adult muscles and embryos.
We use this procedure in laboratory to study skeletal muscle stem cell behaviors such as proliferation, apoptosis, and differentiation, as well as the study abberant muscle stem cell function in diseases such as muscular dystrophy. So let's get started. The basic method for deriving micro explant cultures from skeletal muscles begins with the dissection of target muscles from a freshly cold mouse under aseptic conditions.
In this demonstration, hind limb muscles are dissected, wash isolated muscles through two changes of DF 20 medium and then place into fresh DF 20 medium in a 60 millimeter dish. Using a stereo dissection microscope carefully microdisect the muscles under sterile conditions to exclude fat connective tissue and bone. Next, cut the cleaned muscle pieces into 400 micrometer cubes cubes using jeweler's forceps.
Place the muscle pieces individually into the center 60 wells of a 96 well plate containing 50 microliters of DF 20 per. Well fill the alta wells with saline to prevent drying out of wells containing explan. Check the wells under the microscope and then place the 96 well plate in the 37 degrees Celsius 5%carbon dioxide incubator.
Micro explan attachment and outgrowth is scored after 24 to 48 hours of incubation and thereafter at 48 to 72 hour intervals depending on the rate of growth of the muscles being cultured. Shown here are examples of day two outgrowth from ex explanted adult skeletal muscle and an established ex explant outgrowth with aggregated cultures and high cell density. X explant has scored according to the level of confluence of cells in each individual well, which will be demonstrated later for expansion and isolation of SMS cells.
Outgrowth culture should be individually monitored for cells with a predominantly SMS cell morphology, IE spherical mono nucleate cells with high refractivity which grow in aggregating clusters. Begin this procedure by dissecting E 12.5 embryos from the uterus. E 11.5 to E 17.5 stage embryos can also be dissected in this way.
Place the embryos individually into Petri dishes containing PECM in readiness for detailed micro dissection. Individual embryos are then further dissected to isolate areas rich in skeletal muscle. First, remove the internal organs, dissect out the tail hind and fall limbs, as well as the upper and lower body walls.
Place all dissected tissues into fresh PECM to suss up the embryo. Micro explan cultures microdisect the dissected four limbs, hind limbs and upper and lower body walls to produce small cubes of tissues of equal size. Place these microplants individually into the center 60 wells of a 96 well plate containing 50 microliters of PECM per well.
A minimum of 60 wells containing one x plant per well should be established per embryo studied for culturing embryos. The center 60 wells can be subdivided into regions denoting from where the explan was derived. This design allows 15 wells each containing respectively for limb, upper body wall, hind limb, and lower body wall.
Explan incubate the explan at 37 degrees Celsius and 5%carbon dioxide for three weeks. During the three week incubation, explan are scored on the third, seventh, 14th, and 21st day of culturing using an inverted microscope. Explan are scored according to the level of confluence of the cells in each individual.Well.
This example illustrates the various levels of confluence of E 15.5 primary embryonic explan cultures stained with myth five zero to 14%15 to 24%25 to 49%50 to 74%and 75 to 100%Photographic images of cultures can also be taken once COFLUENT X explan cultures displaying the morphological features of SMS cells can be subculture. Remove culture medium from the selected wells and filter it using a 0.2 micrometer ACR disc. R syringe filter.
Retain the medium for use is conditioned medium to each. Well add 100 microliters of discs diluted one to 10 in PECM. Then return the plate to the 37 degree Celsius incubator for 20 minutes.
After 20 minutes, use a pipette tip to gently scrape the loosened cells from the surface of the well next centrifuge the cell suspension at 1000 times gravity for three minutes to pellet the cells. Cells are diluted by resus, suspending them in two milliliters of PECM and then transferring them into the wells of a fresh 48 well plate. Finally transfer the cell mixture to 48 well plates for further expansion.
To prepare cells for analysis plate display subculture, primary embryonic Splunk cultures in PECM and conditioned medium onto cover slips in 48. Well plates cells are plated at a density of three times 10 to the three cells centimeter square incubator 37 degrees Celsius to allow the cells to attach, which usually take four to six hours for assessment of apoptosis and proliferation. Wash the cells on the cover slips twice in PBS and fix in 4%para formaldehyde for 20 minutes of room temperature after the fixation wash the cover slips for 10 minutes with PBS then stain with 10 micrograms per milliliter of DPI for three minutes.
Wrap the plate with foil wash cover slips once in PBS for five to 10 minutes. After that, remove the cover slip from the well and invert it onto a spot of daco cyto nation. Mounting medium on a glass slide seal the edges of the cover slip with nail varnish for storage.
Wrap the slides in foil and place at minus 20 degrees Celsius for counting. Slides are viewed under fluorescence on an upright microscope and scored for apoptotic and mitotic cells using an IP grae. When explan are carefully implanted from adult skeletal muscle or from embryos, they will begin to generate cells within a few hours to 72 hours of incubation at 37 degrees Celsius.
Depending on their source expansion of the cell population will occur over a period of days for embryo implants or weeks for older skeletal muscle implants to generate high density aggregating SMS cell primary cultures representative results of a successful clonal derivation of an SMS cell are shown here. A single cell isolated into a 96 well plate becomes a colony of single cell origin that eventually results in an established clonal population. MIF five immunohistochemistry is subsequently used to verify SMS cell identity.
Skeletal muscle derived adult stem cells are transplanted into the tibialis anterior muscle of host mice and these cells are identified by beta GC labeling in this image taken at three months post-injection three recently fused beta galacto positive cells stained in blue are observed in a muscle fiber at 14 months post-injection and extensive contribution of btic galacto positive cells in muscle fibers is detected by anti beta galacto antibody stained in brown. A beta galacto positive satellite cell is indicated by the arrow in this image. Staining is absent in the control in which no primary antibody was used when isolated from injected host muscles 12 months post-injection.
The beta galacto positive cells proliferating culture karyotyping of a mouse clonal SMS cell line reveals a normal diploid chromosome complement beta galacto expression in a colony of PD five zero A cells is shown by histo chemistry in vitro analyses of dystrophic embryonic myth five positive myoblasts reveal that these myoblasts are hyper proliferative and prone to apoptosis as shown in this graph, the outgrowth rate of embryonic myoblasts from Muscle X explan culture is increased in both MDX and CAV three knockout mutants when compared to wild type X explan. An example of a MIF five immuno stained explan is shown here. Hyper proliferation of embryonic myoblasts is observed in these MDX and CAV three knockout mutants as determined by KI 67 positive immunoreactivity.
In addition, elevated apoptosis occurs in both these mutants as shown by DPI staining, and this image shows an example of an apoptotic cell indicated by the arrow. We've just shown you how to isolate skeletal muscle stem cells from adults and embryo microdisect explan. When doing this procedure, it's important to remember to take care with the size of your micro dissected explants.
If they're too large, they'll crows and the cells will die. If they're too small, they will not support. Good outgrowth of sales.
Thanks for watching and good luck with your experiments.
