The overall goal of this procedure is to convert fibroblasts into insulin-secreting cells without inducing pluripotency or using retrovital vectors. This method can help answer key questions in the regenerative medicine field and in particular in cell therapy of diabetes. The main advantage of this technique is that we can obtain a switch in the phenotype without inducing a stable pluripotent state in the converting cells and without retroviral vectors.
Demonstrating the procedure will be Georgia Pennarossa, a post-doc in our laboratory. To begin this procedure add 1.5 milliliters of sterile 0.1%porcine gelatin to the 35-millimeter petri dishes and allow coating for two hours at room temperature. Next, wash the human skin biopsies with PBS supplemented with 2%antibiotic antimycotic solution.
Afterward, place the biopsies in a 100-millimeter petri dish and cut them into approximately two cubic millimeter fragments with sterile scalpels. Then, place five to six skin fragments into the pre-coated 35-millimeter petri dish. Add a droplet of fibroblast medium over each fragment and culture them at 37 degrees Celsius in 5%carbon dioxide.
After six days o f incubation, discard the tissue fragments carefully. The fibroblasts should start to grow out of the tissue fragments and begin to form a cell monolayer. In this procedure, culture the fibroblasts at 37 degrees Celsius in 5%carbon dioxide until 80%confluence.
Then plate the cells in new culture dishes. Culture them at 37 degrees Celsius in 5%carbon dioxide and keep the passage ratio between one to two and one to four, depending on growth rate. Next, add 0.1%porcine gelatin to the cell culture dishes and allow coating for two hours.
Remove excess coating solution 10 to 30 minutes prior to plating fibroblasts. Then, remove the fibroblast culture medium from the culture dishes. Wash the cells three times with PBS supplemented with 1%antibiotic antimycotic solution.
Then, add a thin layer of Trypsin-EDTA solution. Incubate it at 37 degrees Celsius until the cell monolayer begins to detach from the bottom of the tissue culture dish and the cells dissociate. After that, dilute the cell suspension with nine parts of fibroblast culture medium in order to neutralize the trypsin action.
Then, count the cells using a counting chamber under the microscope at room temperature. Calculate the volume of fibroblast culture medium required to re-suspend the cells and to obtain the cell concentration of 7.8 times 10 to the 4th fibroblast per square centimeter. Subsequently, centrifuge the cell suspension at 150 g for five minutes at room temperature.
Then, remove the supernatant and re-suspend the cells with a calculated volume of fibroblast culture medium. Afterward, plate the cells on the 0.1%gelatin pre-coated dishes and culture them for 24 hours at 37 degrees Celcius in 5%carbon dioxide. On day zero, immediately prior to use, prepare 5-aza-CR stock solution by dissolving 2.44 milligrams of 5-aza-CR in 10 milliliters of DMEM high glucose medium.
Sterilize it by filtration. Next, dilute one microliter of 5-aza-CR stock solution in one milliliter of fibroblast culture medium. To increase cell plasticity, 24 hours after cell plating remove culture medium from the seeded fibroblasts.
Add one micromolar 5-aza-CR stock solution, and culture for 18 hours at 37 degrees Celsius in 5%carbon dioxide. On day one, prepare fresh HP medium. After incubation with one micromolar 5-aza-CR, remove the medium and wash the cells three times with HP medium to ensure that 5-aza-CR is rinsed away.
Then, incuabate the 5-aza-CR treated fibroblasts with HP medium for three hours at 37 degrees Celsius in 5%carbon dioxide. To monitor the efficiency of 5-aza-CR treatment, check for any morphological changes. Cells should lose the typical elongated morphology of fibroblasts and acquire a smaller round or oval shape with enlarged nuclei.
On days one through six, prepare the pancreatic Basal Medium and Activin A stock solution. Then, culture the 5-aza-CR treated fibroblasts in pancreatic Basal Medium supplemented with Activin A stock solution for six days at 37 degrees Celsius in 5%carbon dioxide and change the medium daily. On days seven through eight, prepare retinoic acid stock solution by adding 16.6 milliliters of DMSO to 50 milligrams of retinoic acid.
Culture the cells in pancreatic Basal Medium supplemented with Activin A stock solution and retinoic acid stock solution for two days at 37 degrees Celsius in 5%carbon dioxide and change the medium daily. On days nine through 36, culture the cells and pancreatic Basal Medium supplemented with ITS, B27, and BFGS stock solution and change the medium daily for the first 15 days and refresh the medium every other day from day 16 onward. Culture the cells at 37 degrees Celsius in 5%carbon dioxide.
This figure shows the morphological changes taking place during the endocrine pancreatic differentiation. After seven days of induction human cells gradually organize in clusters. In response to the addition of retinoic acid, they rearrange in a reticular pattern and cluster in distinguishable aggregates.
These formations progress with time, recruiting cells and aggregating enlarged 3D colonies. Finally, colonies become spherical structures that tend to detach and flowed freely in the culture medium, reminiscent of typical pancreatic islets in vitro. After 36 days of pancreatic induction global DNA methylation levels of human epigenetic converted cells returned to those observed in the untreated fibroblasts.
The co-localization of PDX1 with CPEP is observed at the end of the conversion period while these endocrine pancreatic markers are completely absent in untreated fibroblasts. This graph shows the amount of insulin release from the epigenetic converted cells in response to 20 millimolar D-glucose and 20 millimolar L-glucose exposure. Once mastered, this technique can be done in 38 days if it is performed properly.
After its development, this research paved the way for researchers in the area of regenerative medicine to explore the cell therapy of diabetes in human. After watching this video you should have a good understanding on how to convert fibroblasts into insulin-secreting cells without inducing pluripotency nor using retroviral vectors.
