The overall goal of this procedure is to examine how expression of a gene at different times during differentiation of embryonic stem cells affects hematopoietic differentiation. Following transduction of embryo body, single cell suspension by centrifugation embryo bodies are reformed by hanging drop and cultured to allow for further differentiation into hematopoietic cells. Next single cell suspensions of the reformed embryo bodies are prepared incubated with fluorochrome conjugated antibodies and analyzed on a flow cytometer for hematopoietic differentiation.
Ultimately, this method demonstrates whether expression of a gene during embryonic stem cell differentiation affects hematopoietic development. It is especially useful for demonstrating roles for genes that may only be active following formation of early embryonic tissue. So of the common techniques to express a gene adventurous at different times during embryonic stem cell differentiation require generation of embryonic stem cell lines and inducible express a gene of interest with inducers such as doxycycline.
Generation of these lines can be time consuming. The advantage of this protocol is that can be performed immediately assuming the investigator has the retroviral plasmids for gene expression available. We first heard that this idea about this method when you observed that some genes that were hypothesized would enhance hematopoietic differentiation had an opposite effect in undifferentiated cells.
This suggested that methadone tissues needed to be found to be formed first before this gene had any effect. Begin this protocol with embryonic stem cell derived embryo body or EB formation as described in the text protocol at the desired stage of development. Transfer the EBS from the 10 centimeter Petri dish to a 15 milliliter conical tube.
Wash the plate with five milliliters of phosphate buffered saline or PBS and add the wash to the conical tube. Then pellet the EBS at 315 Gs for five minutes at room temperature. Following centrifugation.
Remove the supernatant and wash the EB cells with five milliliters of PBS following the wash. Centrifuge the cells a second time and remove the supernatant. Next, add one milliliter of thawed cell detachment solution to the pelleted cells.
Place the tubes in a 37 degree Celsius water bath for 30 minutes with occasional agitation. Following incubation, add one milliliter of differentiation media and pipette up and down. Using a two milliliter pipette attached to a P 200 pipette tip.
Place the tube in a 37 degree Celsius water bath again for 30 to 60 minutes after washing the cells. As before resus, suspend the cells in 1.5 milliliters of differentiation media. Then transfer the cell suspension to a six well non tissue culture treated plate.
Add five to 100 microliters of viral supernatant to the cells depending on viral titer. Do not add poly brain to enhance infection as this inhibits the subsequent reformation of ebs. Inoculate the cells with virus by centrifusion at 2, 125 Gs for 90 minutes at room temperature To differentiate the cells first, add two milliliters of differentiation media to each well of infected EB cell suspension.
Then pipette 3.5 milliliters of infected EB cell suspension into a sterile reagent reservoir. For multi pipetters, use the multi pipetter to pipette. 15 to 20 rows of eight 20 microliter drops onto the inverted lid of the 15 centimeter Petri plate.
Add 10 milliliters of sterile PBS or water to the bottom half of the Petri plate. Then invert the lid with the drops and place onto the Petri plate. Ensure that the drops are hanging upside down from the lid with PBS or water below to keep the chamber humidified.
Place the dishes in the 37 degree Celsius tissue culture incubator with 5%carbon dioxide After two days, collect EBS formed in the hanging drops by inverting the lid and washing with 4.5 milliliters of differentiation media. Transfer the media and EBS to a new 10 centimeter non tissue culture Petri plate. Then wash the lid once more with three milliliters of differentiation media and transfer to the 10 centimeter plate.
Harvest the cells for analysis by flow cytometry after a total of eight days of culture, starting from the initial transfer of cells to differentiation media. To prepare cells for flow cytometer analysis. Transfer the cells to a 15 milliliter conical with a 10 milliliter pipette.
Wash the plate with five milliliters of PBS and transfer to the same conical tube After washing the cells as before, add one milliliter of thawed cell detachment solution and place the tubes in a 37 degree Celsius water bath for 30 minutes With occasional agitation, add one milliliter of differentiation media and pipette up and down with a two milliliter pipette. Use a P 200 tip on the end of the pipette to help break up digested EB into a single cell suspension. Place the tube in a 37 degree Celsius water bath again for 60 minutes in order for the integral membrane proteins to recycle to the cell surface.
After pelleting the cells as before, wash the cells with P-B-S-B-S-A, the cells are now ready for incubation with specific fluorescently tagged antibodies. Place a million EB derived cells in a five milliliter round bottom tube. After pelleting the cells at 315 Gs, pour off the supernatant and resuspend the pellet.
In the residual P-B-S-B-S-A solution in the tube for detecting embryonic stem cells, hematopoietic progenitor cells or ESC HPCs, the cells are incubated with fluorescent labeled antibodies anti-US CD 41 R FICO ERYN and anti-US CD one 17 ALLO FICO cyan cyan seven. After determining optimal dilution for labeling cells individually at 100 microliters of the diluted antibody to each sample following a 30 minute incubation on ice in the dark, add two milliliters of P-B-S-B-S-A to the samples and pellet the cells at 315 GS for five minutes. Aspirate the supernatant and resuspend the pellet in 300 microliters of PBS or another isotonic buffer.
Finally, filter resuspended cells through a cell strainer cap to remove large cell aggregates. Before analyzing the cells on a flow cytometer cells are now ready for analysis on the flow cytometer for adjusting flow cytometer compensation settings. Use compensation beads according to manufacturer instructions, then proceed with analysis of cells.
The chime of the EBS between infected and non-infected cells was observed by fluorescence microscopy at day eight of differentiation. The EB is visualized with bright field as well as with fluorescence microscopy in different focal planes and under different magnifications. The density of the EBS often makes the EBS appear to be 100%GFP positive.
However, focusing on different focal planes can reveal the contribution of GFP positive and negative cells to the EBS ESCs. Were infected with MIRN 23 A at the onset of differentiation to determine its effect on blood progenitor differentiation. Hematopoietic progenitor cell generation in chimeric EBS was analyzed by assaying CD 41 and CD one 17 cell surface expression by flow cytometry left hand histogram plots show the GFP minus non-infected cell gates and GFP plus infected cell gates.
The right hand panels show color dot plots of cells positive for CD 41 and CD one 17 expression in the GFP minus and GFP plus gates. CD 41 single positive cells are a pool of primitive and definitive H PCs. Whereas CD 41 and CD one 17 double positive cells contain only definitive HPCs.
A significant increase in the overall population of CD 41 positive HPCs was observed in the infected cells compared to control virus infected cells. The technique is performed over eight days during the protocol. Only three days are labor intensive.
The first day when the initial ESL differentiation are set up, the day when the embryo bodies cell suspicion are infected with later virus and cultured to reform the embryo bodies. And then the final day when the embryo bodies are analyzed. For cytometer Techniques for temporarily expressing genes and differentiating stem cells are critical in the future design of protocols for directing the differentiation of stem cells for use to regenerative medicine.
