This protocol presents multi-stage differentiation of human embryonic stem cells as an effective approach to insulin producing cells with a better yield than previously developed protocols first induce definitive endoderm with active in a and PI three K inhibitor. Then to promote pancreatic progenitor induction, expose cells topamine A and retinoic acid. Next co-culture with endothelial cells to mediate maturation of pancreatic progenitors into insulin expressing cells.
Proceed to functional analyses by immunofluorescence microscopy and QPCR to demonstrate expression of C-peptide and insulin upregulation. The main advantage of this technique over existing methods such as not, is that this method results in higher upregulation of insulin as well as higher yield of cpep that expressing cells Culture. H one human embryonic stem cell colonies to a size of between one and 1.5 millimeters in diameter to the cells CO to 12.
Well plate with one milliliter per well of HESC matrigel diluted in D-M-E-M-F 12 Media in the meantime, aspirate the media from the HSCs and replace the mts are one. Then harvest the cells by scraping. Add them to a 15 milliliter chronicle tube and add fresh MTR one then plate at a split ratio of one to four in the previously matrigel coated wells of a 12 well plate.
Once propagating cells reach one to 1.5 millimeters in diameter. Differentiation is initiated by adding definitive endoderm induction medium containing active in a and the PI three K inhibitor. Wart manin culture for four days.
Replenishing the media daily. Proceed to pancreatic progenitor induction by first culturing in complete D-M-E-M-F 12 containing 0.2 micromolar KAAD, cycl dopamine for 24 hours. Next, exchange media to complete D-M-E-M-F 12 containing 0.2 micromolar of KAAD Cycl dopamine and two micromolar of all trans retinoic acid incubate for three days replenishing media daily culture.
The cells in maturation media for two days prior to performing the final maturation step as a positive control for in vivo maturation of ESC derived pancreatic progenitor cells at the notch inhibitor DAPT prior to splitting. Inspect the endothelial cells under the microscope, then tryps anize and count the cells for the contact co-culture condition. Add 1 million rat heart microvascular endothelial cells to the differentiating cells in complete MCDB 1 31 media.
Also prepare a control culture of co-culture media only with our endothelial cells. Place all treatment conditions in an incubator for six days with media change. Daily collect sample cells from the end of each stage of the differentiation process.
Endo dam, pancreatic progenitor and mature eyelet extract RNAs using nucleus spin E-R-R-N-A to extraction kit according to the manufacturer's instructions. With a smart spec plus spectrophotometer and quartz vet, determine the RNA quality by absorbance ratio 260 nanometers versus 280 nanometers. And also calculate the sample concentrations with 100 nanograms of RNA template per sample.
Perform RT reactions using the impromptu reverse transcription kit. Proceed to set up QPCR using primers shown load reactions onto thermocycler and run QPCR program. Then using the CT values, calculate fold change of target mRNA expression over undifferentiated cells at the end of each stage of differentiation.
Fixed cells in 4%formaldehyde for 15 minutes at room temperature. Then permease with 0.25%Triton X 100 for 10 minutes to block non-specific staining. Add 10%donkey serum in 0.05%TX for 30 minutes.
Next, add primary antibody in blocking buffer and incubate overnight at four degrees Celsius. Wash samples with 0.05%Triton X for five minutes. Then add the secondary antibody diluted in blocking, buffer and incubate for one hour of room temperature in the dark.
Perform three five minute washes of 0.05%x to stay nuclei at horse stain at one to 1000. Dilution in PBS incubate for five minutes, followed by three washes with PBS. Acquire images of the fixed and stain cells using inverted microscope and imaging software.
Addition of active in a and wart manin to undifferentiated human embryonic stem cells for four days induces definitive endoderm. The Q-R-T-P-C-R analysis confirmed presence of the expected definitive endoderm markers. SOX 17, CXCR four, and Fox A two.
Immuno staining for SOX 17 in green shows colocalization with the blue DPI stained nucleus here. Differentiation to pancreatic progenitor cells after addition of cyclo, amine and rettino acid was confirmed by Q-R-T-P-C-R of pancreatic progenitor markers and also immuno staining for PDX one. At the final stage of differentiation, contact co-culture with rat heart microvascular endothelial cells strongly induced upregulation of insulin expression in the HESC derived pancreatic progenitor cells.
L co cultures with AC LDL labeled RHM VEC show regions where ECS attached to the plate. If there are empty spaces, other RHM VEC can be found in direct contact with the differentiating ESC as expected. Immuno staining of RHM VEC demonstrates no detectable insulin expression.
Eptide staining is also a useful marker for cells differentiated using co-culture method After its development. This technical allowed investigation of cell cell interactions in the process of stem cell differentiation, which evidently increases the functionality of the mature phenotype.
