The overall goal of this procedure is to isolate naive multipotent, skin derived precursor cells from primary human fibroblast cultures. First, establish a primary fibroblast culture and select for naive skin derived precursor cells based on resistance to stress conditions like temperature variations and nutriment depletion. Next culture, the cells to form spheres under controlled growth condition continue to grow and expand the spheres cultures for several weeks and demonstrate that these precursor cells share similar properties to adult stem cells.
Ultimately, SKP cells can be directed to differentiate into smooth muscle cells. I first had the idea of this method when I observed that in frozen OTs of primary fibroblast structures that experience stress because of temperature variation during storage or shipment, a few cells remain competent to go. This suggested that a small number of cells were resistant to stress and might be dermal stem cells demonstrating the procedure will be relevant.
A graduate student in my lab who has been involved in the development of the SKP isolation method from human fibroblast cultures For the SKP isolation culture, fibroblasts to population doublings of 20 to 35 at a co fluency of 80%Wash the cells once with PBS and incubate with two milliliters of trypsin solution for one hour at 37 degrees Celsius. Harvest the cells with five milliliters of PBS and transfer the cell suspension to a 15 milliliter Falcon tube, then incubate at four degrees Celsius for 24 hours. Centrifuge the suspension at 1200 RRP M for five minutes.
Re suspend the cell pellet directly in four milliliters of SKP growth medium and set a culture to grow at 37 degrees Celsius, 5%carbon dioxide every day. Shake the flask vigorously to avoid cells adhesion if necessary. Use a two milliliter sterile pipette to detach the adherence cells and transfer the culture to a new flask.
After three to four days when the first spheres start to build, allow the spheres to sediment and change half of the medium with the freshly prepared SKP growth medium containing two x growth factors culture. The cells replenishing the medium every three days to passage spheres of about 200 microns. Break the spheres by vigorous pipetting, then split the culture into 2 25 centimeter squared flasks and replenish the medium by day 16 to 21.
Collect the spheres for screening of stem cell markers. Pellet the cultures by centrifugation. Then re suspend the spheres in a small volume of PBS with a deco pen.
Draw two circles of approximately 0.5 micrometers in diameter on microscope slides at 50 microliters of sphere suspension into the circles. Verify the presence of spheres in each drop by microscopy. Then place the samples to dry for immunofluorescent staining.
Fix the slides with pre-cool 100%methanol for 10 minutes. Wash once with PBS.Next. Block the samples for at least one hour.
Incubate with primary antibody solution for two hours at room temperature. After washing three times with blocking buffer. Incubate with secondary antibody for one hour at room temperature.
Then wash three times with blocking buffer and three times with PBS. Mount the slides with fecta shield mounting medium. Then analyze the samples for expression of stem cell markers by immunofluorescence microscopy First pellet, approximately two to three milliliters of sphere cultures from day 18 to 21 by centrifugation and aspirate off all the medium.
Isolate the RNA using the RN easy mini kit. Then assess the RNA purity by spectrometry and also acro electrophoresis. Next synthesize CD NA using the total cellular RNA as template.
Now to validate the stem cell markers by realtime PCR, use a power cyber green PCR master mix with a concentration of 375 nano molar of each primer and 50 nanograms of template. In a 20 microliter reaction volume, remember to include the gap DH measurement as endogenous control for the amplification. Use an initial denaturation at 95 degrees Celsius for two and a half minutes, followed by 40 cycles at 95 degrees Celsius for five seconds and 60 degrees Celsius for 20 seconds.
Analyze the run as described by VAC and Schmidt in 2001. Plate the spheres from day 21 to 26 into six. Well culture dishes in SKP medium culture cells for 72 hours so that they adhere and outgrow from the spheres.
In order to initiate smooth muscle cell differentiation. Replace the medium with SMC differentiation, medium culture for three to four weeks. Replenishing the medium every three to four days.
Now to screen for SMC markers by immunohistochemistry. Fix the cells with 4%paraform aldehyde solution in PBS for 10 minutes. Then perme the cells with PBS containing 0.3%Triton X 100 for 30 minutes.
Next, add primary antibodies against alpha smooth muscle actin or pontin incubate for one hour at room temperature process with secondary antibody. Mounting the slide as described earlier, this population of cells derived from primary dermal fibroblast cultures were selectively expanded to generate S KP spheres under controlled growth conditions consisting of e GF and FGF two immunohistochemistry analyses show that these SKP cells express neuro crest and neuron stem cell marker neston, as well as the embryonic stem cell transcription factors, NANOG and OCT four and the pluripotent cell surface marker TG 30. Similarly to the SKP derived directly from skin biopsies, the NEIN positive signal resides in the cytoplasm while the cell surface marker of pluripotent human embryonic stem cells.
TG 30 has a positive cytoplasmic membrane. Staining T four shows a punctuated nuclear signal and nano displays a more diffused nuclear signal. Real-time PCR confirms the presence of transcripts for the multipotent stem cell markers Nest 10 and OCT four in RNA preparation isolated from 18 day old SKP cultured spheres.
To further test the multi potency of the SKP derived from fibroblast cultures, we induce these cells to differentiate into smooth muscle cells. After a three weeks induction in medium containing the growth factors, P-D-G-F-P-B, and TGF beta one immunochemistry confirmed the expression of the smooth muscle cell markers. After watching this video, you should have a good understanding of how to isolate derma stem cells from primary fibroblast cultures.
We hope that this approach will open new possibilities for your research.
