The overall goal of the following experiment is to induce and assess reprogramming of somatic cells towards pluripotency in their in vivo microenvironment. This is achieved by loading a syringe with plasmid DNAs encoding the Yamanaka factors and hydrodynamically injecting them into the tail vein of A BC mouse as a second step. Primary hepatocytes are isolated from liver tissues and relevant gene and protein expression levels are studied by real-time Q-R-T-P-C-R and flow cytometry.
Next whole liver tissues are sectioned and stained by different techniques in order to confirm the reprogramming of the tissue and evaluate the toxicity of the procedure. Results are obtained that show differences in gene and protein expression of reprogramming, pluripotency and hepatocyte specific markers. Furthermore, the safety of the approach is also validated with biochemical and histological evidence.
The main advantage of reprogramming somatic cells in vivo is that it happens fast, efficiently, and transiently. This method can help answer key questions in the reprogramming field. For example, the outcomes of inducing pluripotency in adult in vivo.
To begin anesthetize, a six week old female bulby mouse. Then prepare a plasmid DNA solution ENC coding the Yamanaka factors and a saline control as described in the accompanying text protocol For the transfection to be successful, inject the entire dose in no more than five to seven seconds at predetermined time points following the injection. Isolate the primary hepatocytes by exposing the thoracic cavity and perfusing the liver with prewarm HBSS buffer and then liver digest medium perfuse at a flow rate of 0.6 liters per minute for 15 minutes until the liver becomes swollen and loose.
Then remove the liver by forceps after cutting the falor and coronary ligaments. For more details on the isolation of primary hepatic cells, see the steps in this additional JoVE video. Next, wash the liver with hepatocyte wash medium while passing it through a 100 micron cell strainer.
To obtain a cell suspension, collect the cell suspension in 50 milliliter tubes and adjust the volume to 15 milliliters with hepatocyte wash medium. Then centrifuge the cell suspension at 50 G for four minutes at four degrees Celsius. Discard the supernatant and suspend the cell pellet.
In 10 milliliters of hepatocyte wash medium, the cell pellet should be suspended carefully as hepatocytes are very fragile cells. Repeat the centrifugation procedure for a total of three times and suspend the final pellet in 10 milliliters of hepatocyte wash medium. Then estimate the cell number using a hemo cytometer and an optical microscope to study the gene expression of isolated hepatocytes.
Start by centrifuging two times 10 to the sixth cells at 300 times G and discard the snat. Then extract the RNA from the cells with a spin column kit by following the instructions of the supplier. Next, use one microgram of the extracted RNA from the treatment and control groups to perform two-step Q-R-T-P-C-R in order to look for changes in the levels of reprogramming pluripotency and hepatocyte markers.
To analyze the enhanced pluripotency of cells at the protein level, take the isolated hepatocytes and adjust the density of the cells to one times 10 to the seventh cells per milliliter. For flow cytometry, prepare 100 microliter aliquots of the cell suspension and then fix them in 100 microliters of BD cyto. Fix fixation buffer for 20 minutes at room temperature following fixation, pellet the cells and resus, suspend them in 100 microliters of one X BD permeation wash buffer incubate for 10 minutes, add room temperature.
Next, incubate the cells with primary antibodies for transcription factors expressed in pluripotent cells for 30 minutes. This includes either anti mouse T four per CP SCI 5.5 or anti-US nag pe. Be sure to also include negative and isotype controls.
Then analyze the stained cells in the flow cytometer with per CP sci 5.5 and PE channels four days following injection of the plasmid DNA cryo section, the liver as described in the accompanying text protocol. Then incubate methanol fixed sections in blocking buffer at room temperature for one hour, followed by two wash steps for five minutes each with wash buffer. Next, add anti T four anti socks two and anti nag primary antibodies onto separate slides, making sure to completely cover the liver tissue.
Cover the samples to preventive aberration and incubate the sections overnight at four degrees Celsius. The next day, wash the sections three times for five minutes each with wash buffer. Then incubate them for 1.5 hours at room temperature with their respective secondary antibodies following incubation.
Wash the liver sections three times for five minutes each with PBS. Mount them in dappy anti fade medium and protect them with cover slips. Then visualize the slides with an epi fluorescence microscope.
Warm the cryo sections to room temperature for 15 minutes and fix them with 4%paraform aldehyde for two minutes. Then air dry the slides for 30 minutes. Once dry, incubate the sections with B-C-I-P-N-B-T liquid substrate at 37 degrees Celsius for 30 minutes.
During this time, the solution will react with alkaline phosphatase in the tissue to form an insoluble precipitate visible by microscopy. After 30 minutes, wash the slides with distilled water and mount them with aqueous mounting media. Then capture images using light microscopy on days 4, 8 12, and 120.
After transfection, collect 300 microliters of blood directly from the heart ventricle. Keep the blood on ice while the different samples are being collected to keep it from clotting too early. Once all the samples are collected, incubate them for 30 minutes at 37 degrees Celsius or until the blood clots.
Then centrifuge the samples at 2000 G for five minutes and carefully pipette the serum into a new einor tube. Then analyze the serum levels for albumin and the liver enzymes, A-L-T-A-S-T-A-L-P and GLDH using a spectrophotometer. In addition to screening the blood in order to assess liver toxicity, also prepare paraffin embedded tissue sections as described in the accompanying text protocol.
Then stain the tissue with H and D or PIS staining and analyze the images captured. Light microscopy following hydrodynamic tail vein injection of the plasmids, a significant increase in gene expression of the transfected reprogramming factors. T three four socks two and cmic at the mRNA level was observed on day two after injection.
The expression of these factors decreases over time after injection. The endogenous pluripotency markers, nano g ECAT one and REX one, or also significantly upregulated compared to those in hepatocytes from saline injected animals on day two and four after injection and were back to baseline levels from day eight onward. At the same time, de-differentiation of the hepatocyte population was confirmed by the downregulation of hepatocyte specific genes, A-L-B-A-A-T, and TRF that were statistically significant on day four and reached baseline levels from day eight onward.
Also, the expression of T three four and nag at the protein level was investigated by flow cytometry as shown here. Only OC three four is expressed on day one. After HTV injection, while for the expression of the endogenous pluripotency marker nano G, it was necessary to wait until day four.
The occurrence of in vivo cell reprogramming was further confirmed by immunohistochemical analysis of liver tissues. Positive cells for all markers and enzymatic activity are reproducibly found in the liver tissues from KSM injected animals, but not in the saline injected controls. H and e and PAS staining show transient but not severe signs of tissue damage which dissipated after day two.
No formation of teratomas or any sign of dysplasia or morphological alterations was observed for the period of the study. There were also no hepatic functional abnormalities throughout the course of the study for any of the animals as confirmed by albumin and liver enzyme levels shown here. This study provides proof of principle evidence of in vivo cell reprogramming towards pluripotency.
This happened rapidly, transitively and efficiently after the administration of the Yamanaka factors. We hypothesized that in vivo reprogramming can be achieved in other tissues other than the liver provided that adequate gene. Liver vectors are designed to transfect the target tissue.
Furthermore, with improved protocols and technologies, it might be possible to extract the in vivo program cells to be used for further studies.
