プラセンタから造血幹細胞の単離と解析

Hi, I am Mikkola and welcome to my laboratory. The primary focus of our lab is to investigate hematopoietic stem cell development. During embryogenesis, we have identified the placenta as a major hematopoietic organ and found that hematopoietic stem cells are both generated and expanded in the placenta in unique microenvironmental niches.

As such, the placenta has a tremendous value as a source of hematopoietic stem cells during their emergence and initial expansion. Today the members of my lab will show the experimental techniques required for isolation of hematopoietic stem cells from the most placenta and visualizing HCS in their unique microenvironmental niches. Hi, I'm Chriss Geka from the laboratory of Dr.Hannah Mola in the Department of Molecular Cell and developmental biology at UCLA.

Today I'll be showing you how to dissect mouse placenta and how to prepare single cell suspension of placental tissue for fax analysis for fetal hematopoietic stem cells. And I'm Katrina Rhodes, and I'll be showing you how to prepare placental tissue for immunohistochemistry. So let's get started.

To begin, embryos must first be isolated from pregnant dams. Animals are euthanized according to approved procedures. In our case, we're going to use a lethal dose of the anesthetic isof fluorine.

First, spray the belly with ethanol and make a small incision with a pair of scissors. Tear away the skin with both hands to uncover the abdomen and cut open the peritoneum with forceps. Pick up the uterine horns and collect them using scissors at each distal end.

Place the uterine horns in a Petri dish filled with PBS and place on ice. Also, remember to wash several times with PBS before isolating the placenta with two forceps. Begin carefully peeling away the endometrial tissue surrounding the embryo concepts.

These embryos are from embryonic day 12.5, which is the developmental age in which hematopoietic stem cells or HSCs are at their peak in the placenta. One should be careful not to puncture or otherwise inflict structural damage to the embryos as this will complicate subsequent dissection steps. Place the isolated concepts in the new petro dish with PBS on ice and continue until all the concepts have been freed from the endometrium.

Transfer one conceptus to the new Petri dish with PBS placed under the microscope. And with two forceps, peel the deidra away from the G placenta. Remove as much of the deidra as possible as deidra cells will interfere.

With single cell suspension and flow cytometry. A smooth continuous peeling motion is recommended to achieve the best results. Of note, when working with younger embryos like these embryonic day 10.5 or E 10.5 embryo, the deidra will surround the entire conceptus.

Unlike E 12.5 embryos at E 10.5, HSCs are just beginning to emerge within the placenta. Notice the considerable size difference between E 10.5 and E 12.5 embryos. Cut the yolk sac away from the placenta at the junction between the two organs, and gently pull the yolk sac and beline vessels away from the placenta.

Care should be taken not to disrupt the choic plate of the placenta, especially with early embryos. Carefully holding the CHO plate of the placenta with one pair of forceps in the umbilical cord with another. Prove the umbilical cord and attached embryo away from the placenta.

Remove excess giant cell tissue at the edges of the placenta In order to minimize cell clumping during preparation of single cell suspension, collect the placenta in a suitable container such as a 15 ML Falcon tube with PBS and 5%FCS and place it on ice. Do this for all placenta dissected. At this point, you can either proceed with the preparation of a single cell suspension for flow cytometry functional assays such as in vitro culture or transplantation, or prepare whole placenta for tissue fixation and fixed frozen block embedding for eventual immunohistochemistry.

We will now show you how to generate a single cell suspension from placenta for fax analysis. First, prepare a 0.1%collagenase solution in PBS with 10%FCS and 1%penicillin streptomycin solution. Add an appropriate volume of collagenase solution to the placenta depending on how many you have a volume that is twice the placenta volume in between two to five milliliters is recommended.

Use a 16 gauge needle fitted on a five ml syringe to mechanically disrupt disruptive tissue by packaging the collagenase solution and placenta through the needle three times. Repeat with an eight gauge needle. Place the sample in a 37 degree Celsius 5%CO2 incubator for 45 minutes after the 45 minute incubation.

In collagenase passage, the cell solution through a 20 gauge needle and incubate for an additional 45 minutes in 37 degrees after the one and a half hour total incubation in collagenase passage, the cell solution through 22 gauge and 25 gauge needles three times with each needle. Now filter the sample through a 50 micrometer filter attached to a new 15 ML Falcon tube wash with PBS plus 5%FCS and centrifuge at four degrees Celsius for five minutes at 300 gs. At this point, we have a single cell suspension suitable for fax analysis.

I will show you how to prepare placental tissue for immunohisto chemistry. Immunohistochemistry of placental tissue can be used to localize hematopoietic stem cells within the placental microenvironment. Immediately after isolating the placenta from each embryo, they're placed in cold one XPBS, usually a 96 well plate works best for young placentas embryonic day 8.5 to 11.5, but with older placenta E 12.5 and older, A 24 well plate may be more convenient.

Each placenta is transferred into a new well filled with freshly thawed 4%PARALDEHYDE or PFA For the E 12.5 day placenta. The tissue is moved to a new well by hand. But for the E 10.5 placenta, you can remove the PBS carefully with a pipette and add the PFA.

It is important that the placenta is fully immersed for two to four hours at four degrees C, depending on the age and size of the tissue. Then transfer the tissue into 30%sucrose. Or if the tissues are very small and fragile, aspirate the PFA and add the sucrose directly.

At this stage, the placenta will be floating in solution. Let the tissue stay overnight at four degrees C.After the overnight step in 30%sucrose, the tissues should ultimately sink to the bottom of the well indicative of proper cryopreservation. Next, remove half of the sucrose solution and replace the volume with OCT.

Place the tissue back in four degrees C for one to two hours. This facilitates OCT penetration. Finally, the tissue will be transferred to a hundred percent OCT for one hour, after which it can be embedded.

Label plastic molds with appropriate tissue identification. Pull the tissue out of the OCT solution and cut the placenta in half by orienting the disc shaped placenta with the umbilical cord side facing down and carefully slicing with the clean razor blade, making sure to move the blade back. And fourth, before releasing pressure to ensure an even cut.

The placenta is cut in half to achieve the best orientation for later visualization of the placental hematopoietic stem cells. Then place one placenta half at the bottom of the mold with the cut edge and contact with the bottom surface of the mold. Repeat with a new mold for the other half.

For E 10.5 placentas, both halves may be placed into the same mold using the same technique. When pouring OCT into the mold, it can be difficult to keep the placenta in the proper orientation. Secure the placenta in place by holding the tissue half with forceps for E 10.5 placenta.

Hold one tissue half with forceps and rest the other tissue half against the outside of the forceps. Then slowly pour OCT solution as described for E 12.5. Once the mold is filled with OCT, quickly place the mold onto dry ice.

The OCT will turn white flash freezing the tissue, place the mold into a small plastic bag and immediately store in a minus a DC freezer. Here is a representative section of an E 10.5 placenta. It is important that your sections include the chorionic plate shown at the bottom of the image and the placental labyrinth, the region of the placenta, which facilitates fetal maternal exchange shown in the middle.

This section was stained with a CD 31 antibody, which has marked the endothelium red. The trophoblasts shown in brown are marked by cytokeratin. The labyrinth harbors both fetal vasculature vessels marked in red, as well as maternal blood spaces lined by the brown trophoblasts.

The blue cells are CD 41 positive nascent hematopoietic stem and progenitor cells notice that they localize to the fetal vasculature. We've just shown you how to dissect and prepare a single cell suspension of mass placenta and also how to identify placental hematopoietic stem cells by flow cytometry. We've also shown you how to prepare sections of placental tissue for the visualization of hematopoietic stem cells by immunohistochemistry.

It is important to pay attention to a couple of critical parameters when performing these procedures. When doing the placental dissection, it is important to work quickly and to keep embryos and solutions so nice as much as possible in order to maintain high cellular viability for the preparation of single cell suspension. The degree of mechanical dissociation prior to and the incubation time during the enzymatic treatment are the most critical factors affecting viability and cell heal.

And lastly, when doing immunohistochemistry over fixation can pose problems for some antigens and on the other hand, under fixation can lead to tissue destruction. So that's it. Thank you for watching and good luck with your experiments.