Placenta research 101

Quick primer for new lab members. Placenta genetics, cell lines, and vocabulary you'll hear in lab meetings.

Placenta & fetus - they develop from the same 1st cell

The placenta and the fetus both come from the fertilized egg.

• 1 cell = zygote (fertilized egg)
• 16 cells = morula (a solid ball of cells, 3-4 days after fertilization)
• 32 up to 150 cells = blastocyst (a hollow ball of cells, starts 5 days after fertilization)

After blastocyst stage, the cells rearrange. The outer layer, called the trophectoderm, develops into the placenta. The inside cells, called the inner cell mass, become the fetus.

Placenta genetics = fetal genetics

This fact is the basis for chorionic villus sampling (CVS), a prenatal diagnostic test performed in late 1st trimester.

Exception: there are rare cases of mutations or genetic abnormalities in the placenta that aren't in the fetus, likely because they occurred in the trophectoderm cells (which become the placenta) and not in the cells that developed into the fetus, or possibly because DNA repair pathways or programmed cell death removed the genetic abnormality. This phenomena is called "confined placental mosaicism."

CVS = chorionic villus sampling

CVS is a prenatal diagnostic test performed in late 1st trimester, usually weeks 10-13 of gestation, to learn about fetal genetics without actually disturbing the fetus. The outer layer of the placenta has chorionic villi (it's a fuzzy outer surface, not smooth). Doctors take a biopsy of the chorionic villi and use it as a source of cells. CVS can be used for karyotype testing, FISH tests, and other genetic tests.

CVS is considered an invasive prenatal diagnostic test. It has a higher risk of miscarriages (approximately 0.07%) compared to non-invasive tests such as blood tests, urine tests, cheek swabs, or ultrasounds. Because of this, CVS is typically only recommended for women who have a higher risk of genetically abnormal pregnancies (e.g. due to advanced maternal age), pregnancies where the parents are carriers for genetic disorders (e.g. cystic fibrosis), or pregnancies that had ambiguous non-invasive test results. One of the goals of our research is improving the availability and reliability of non-invasive tests.

Advanced maternal age (AMA) is >35 years old. 

Placentation = placenta development during early pregnancy

The establishment of the placenta occurs in 1st trimester. The pathogenesis (disease development) of many pregnancy complications is thought to involve differences in placentation, even though the complications may not be apparent or diagnosed until 2nd or 3rd trimester. One of the goals of our research is understanding placenta gene expression during 1st trimester so we can better understand what leads to pregnancy complications.

Decidua = the endometrium during pregnancy

• The endometrium is the lining of the uterus. 
• Hormones during pregnancy transform the endometrium into decidua. In the lab, endometrial cell lines can be treated with hormones to undergo decidualization.
• Maternal-fetal communication (secreted proteins & receptors & hormones) in early pregnancy allows fetal cells to find the decidua and attach, beginning placentation.
  
  

Primary cells vs cell lines

Primary cells 

• Primary cells are cells taken directly from original samples (e.g. tissue, blood) recently. They are the most like their original cells when freshly collected, but will eventually start changing gene expression and phenotype in culture.
• Not immortal
• Will not survive in culture very long, or at all
• Require specific culture conditions to survive: temperature, oxygen and CO₂ levels, growth media, pH, supplemental growth factors, ideal cell density, etc.
• Cell density is especially important for mammalian cells (compared to bacterial cells) because mammalian cells secrete and detect growth factors for cell-cell communication to regulate their growth. They will die if they are too lonely or too crowded.   
• Mammalian cells are also less resilient to environmental shocks, compared to bacterial cells. Forgetting to change a cell flask media or passage cells before a holiday weekend can lead to overcrowding resulting in media imbalances (pH changes, growth factor changes, nutrient depletion) that affect cell growth even after the cell density and media conditions are improved. 
• Historically, finding the correct conditions for growing human cells in culture was difficult. The first successfully cultured human cell line was named HeLa, grown from cervical cancer cells taken without informed consent from Henrietta Lacks, before current day biomedical ethics laws. 
• CVS tissue samples are originally mostly epithelial and fibroblast cells, but the epithelial cells (trophoblast cells) senesce or differentiate within a few days or weeks in culture. Fibroblast cells survive for a few months in culture if conditions are good, but eventually also senesce.

Cell lines

• Cell lines come from ("are derived from") primary cells transformed in some way
• Typically refer to immortalized cells, meaning cells with altered genetics that prevent senescence, either due to naturally occurring diseases (cancer) or biotechnology (introduction of SV40 genes or addition of telomerase). Immortalized cells are more resistant to senescence and less picky about growth conditions compared to primary cells. 
• May also refer to induced pluripotent stem cell (iPSC) clonal lines. These cells are derived from primary cells that were treated with various chemicals and growth conditions to revert back to a stem cell state, then treated with other cell conditions to differentiate to specific cell types. 
• iPSCs are not typically immortalized. 
• Beware: cell culture causes evolutionary pressures. The more generations that cells are in culture, the more likely they are to evolve for the cell culture environment. Cell lines derived from male samples have been found to drop chromosome Y and undergo other genomic changes.  
• Before studying specific genes in cell lines, check if your gene is expressed in the cell lines by qRT-PCR. 
  
  

Trophoblast cells = placenta-specific cell types

In early cell development, the blastocyst meets the maternal decidua. The outer cells of the blastocyst (trophectoderm cells) begin placentation.

• Trophectoderm (TE) is not really part of the placenta organ. It exists during the blastocyst stages of early human development. It is a stem cell type that develops into the placenta. 
• Cytotrophoblasts (CTBs) develop from trophectoderm cells. They are pluripotent stem cells. Their job is to make two other types of trophoblasts: syncytiotrophoblasts and extravillous trophoblasts. Cytotrophoblasts are notoriously difficult to keep alive in cell culture. They differentiate into other trophoblasts or senesce (stop dividing and die).
• Syncytiotrophoblasts (STBs) are fused cells that act like a wall. Multiple cytotrophoblast cells join together into multi-nuclear syncytiotrophblast cells. They form a cell barrier between the fetus and the outside environment. They physically protect the fetus from pathogens. They also produce hormones that help quiet the maternal immune system (to prevent miscarriages) and sustain the pregnancy.
• Extravillous trophoblasts (EVTs) are the cells that migrate and invade maternal decidua and maternal spiral arteries. They detect oxygen. They bring food to the placenta and the fetus. Over- or under-invasion of extravillous trophoblasts into maternal tissue can lead to pregnancy complications later, such as preeclampsia (associated with under-invasion) or placenta previa (associated with over-invasion).

All trophoblasts are epithelial cell types, and they express the epithelial marker CK7 (also called KTR7, cytokeratin 7, or keratin 7). Since trophoblasts are the only epithelial cell types within placenta tissue, CK7 is often called a "trophoblast marker" in placenta papers, but beware that all epithelial cells express CK7.

Cell lines for trophoblast research

• HTR8/SVneo ("HTR8") = human extravillous trophoblast cell line, originally from 1st trimester extravillous trophoblast cells that were immortalized in lab. Female. It is used to study trophoblast cell migration and invasion.
• BeWo = human trophoblast-like cell line, originally from choriocarcinoma primary cells. Male. Cell fusion can be induced using drugs like forskolin. It is used to model cytotrophoblast development into syncytiotrophoblast cells. BeWo cells are the oldest cell line model used to study trophoblast cell function.
• JEG3 = human trophoblast-like cell line, originally from a sub-population of BeWo cells. Male. They act more like extravillous trophoblasts and are also used to study migration and invasion.
• Stem cells. There is no good immortalized cell model for cytotrophoblasts (the placental stem cells). Primary cytotrophoblast cells do not survive long in cell culture. They differentiate further and/or go into senescence (cell aging, cell division stops, eventually cell death). The best cell models for early cytotrophoblast development are induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs), which can be carefully grown in various media to induce differentiation into cytotrophoblasts, and then quickly further differentiation. It is difficult to keep cytotrophoblasts as cytotrophoblasts.
• iPSC = induced pluripotent stem cells. These are often derived from skin fibroblast cells.
  hESC = human embryonic stem cells.
• T-HESC = human endometrial stromal cells (not the same as hESC). These are used as a model for the maternal endometrium and decidua.

Cell culture reagents

• General notes: https://cellculturedish.com/cell-culture-basics-stem-cell-media-the-what-and-why/
• Media = the solution used to grow cells in culture (in flasks or petri dishes). Media contains nutrients, buffers to maintain proper pH, and various additives to keep cells alive.
• RPMI 1640 ("RPMI") with added HEPES buffer and L-glutamine (Gibco) - used for HTR8/SVneo cells
• DMEM - used for JEG-3 cells
• DMEM:F12 (ATCC) - a mix of two recipes, also used for JEG-3 cells
• F12 (ATCC) - another media recipe 
• MEM-alpha (Gibco or ATCC) - used to spin down cells for certain experiments. It is a more general and cheaper media.
  
• Chang medium - a more expensive media that we use for primary villi cells. It contains 16% FBS and growth factors for chorionic villi. Do not waste this expensive media. 
• Anti/anti = antifungal/antibiotic. It is used as an additive for primary cells, tissue that will be cultured, and newly thawed cell vials that need extra time to recover from being frozen. We do not use it longer than 1 week because it makes the cells a bit unhealthy (too strong).
• Charcoal-stripped FBS = fetal bovine serum filtered through charcoal to remove various impurities. It is used for hormone experiments and cell migration experiments.
• DPBS buffer = Dulbecco's phosphate buffered saline. It refers to a specific PBS recipe common in cell culture. When human cell culture protocols say "PBS", they usually mean DPBS.
• FBS = fetal bovine serum. It is used to keep cells alive in culture. It contains growth hormones and growth factors. For most media preparations, we add 10% FBS by volume.
• 50 ml of RPMI + 10% FBS + 1% P/S = 44.5 ml RPMI media + 5 ml FBS stock + 0.5 ml 100x penicillin/streptomycin stock.
• The proteins and hormones that make FBS valuable can break down if the FBS is frozen & thawed too many times or left in a 37C water bath for too long. Store it long-term at -20C, then store FBS in the fridge at 4C when you are actively using it. Do not re-freeze it. Warm it in the 37C water bath only 10-15 minutes before use.
• PBS buffer = phosphate buffered saline, usually at the physiological pH of 7.4. 
• We use PBS buffer in cell culture to wash cells without shocking them with different osmotic pressures or different pH.
• In our lab, protocols saying "PBS buffer" refer to pre-made "1x DPBS buffer". Do not use the powdered PBS because that requires titrating to pH the buffer and then sterile filtration which ends up more laborious and costly.
• Pen/strep (P/S) =  penicillin and streptomycin, antibiotics that we use instead of anti/anti once the cells are growing well. They are less harsh than anti/anti. Even so, we stop using them right before cell behavior experiments (e.g. migration assays) since they can affect cell behavior.
• Phenol red = the red dye in cell culture media that we use as a pH indicator. 
• Red/pink is good, indicative of fresh media and physiological pH conditions.
• Orange is old media. The cells are depleting nutrients and accumulating cell waste (affecting the pH). Change the media and reduce cell density as usual.  
• Yellow orange is way too acidic - the cells are dying. Change the media and reduce cell density more often. 
• Bright neon yellow indicates acidity levels that can't be reached by mammalian cells. It indicates a bacterial contamination. Do not open the flask inside the biosafety cabinet. Add bleach, wait 30 minutes, and discard. Do not risk spreading the bacterial contamination. Inform the lab because we may need to discard other flasks and possibly thaw a new cell vial to start over.
• Trypsin = a protease enzyme that breaks protein-protein bonds required for cell attachment
• Trypsin stocks are thawed once, then aliquot under sterile conditions into smaller tubes. Store the stocks and extra aliquots at -20C long term, and keep an aliquot at 4C while in use. 
• Don't repeatedly freeze and thaw trypsin because this damages enzyme activity.
• Don't leave trypsin in a 37C water bath for too long because this damages enzyme activity.
• What concentration of trypsin are you using? 
• 0.25% trypsin for detaching cells from the flask (cell culture)
• Up to 2.5% trypsin for dissociating cells in tissue samples (tissue dissociation) - this is 10x as concentrated and much harsher! Never use 2.5% trypsin for regular cell culture.
• Wash cells with PBS buffer before adding trypsin. This is because the FBS serum in media inactivates trypsin. Remove the PBS buffer before adding trypsin.
• Do not rely on protocol incubation times (e.g. "5 minutes") because newer and older batches of trypsin have different levels of enzymatic activity. 
• Incubate cells with trypsin at room temperature or 37C and check flasks under the microscope to decide when to stop the incubation. What is the goal? If it's cell detachment, then rock the flask back and forth and visually check for cell detachment under the microscope every few minutes.
• Do not over-incubate cells with trypsin. Trypsin first causes cell-cell and cell-flask detachment, but eventually trypsin damages the cell membrane itself. Keep the cells healthy by avoiding prolonged incubation in trypsin.
• Inactivate the trypsin by adding media containing FBS. Finish cell-cell dissociation by pipetting up and down with a serological pipette.

Hormones

• Estradiol, estrogen. Detected by estrogen receptors alpha and beta, encoded by genes ESR1 and ESR2. Once the placenta is established, the placenta itself produces estradiol. 
• HTR8 cells don't express ESR1, and only weakly express ESR2.
• Progesterone. Detected by the classical progesterone receptor, encoded by gene PGR (also called PR). It also binds membrane-bound progesterone receptors encoded by PGRMC1 and PGRMC2, as well as other non-classical receptors. Progesterone is sometimes administered before or during pregnancy. It helps "quiet" some immune response in the uterus and may prevent miscarriages and preterm delivery. Once the placenta is established, the placenta itself produces progesterone.
• HTR8 cells don't express PGR but do express other progesterone receptors.
• Testosterone. Detected by the androgen receptor, encoded by gene AR. Not produced by the placenta. Any testosterone during pregnancy is produced by the mother or the fetus as far as we know.
• Phenol red. Not a human hormone, but has estrogen-like effects. For hormone experiments, use phenol red-free media. It should be colorless, not pink.