Intro to human blood components, cell types, and cell markers

Blood components as separated by density gradient

1. Plasma: yellow layer on top, 55% of blood volume

2. Buffy coat (leukocytes and platelets): white layer in the middle, <1% of blood volume

• White blood cells (leukocytes) = granulocytes (neutrophils, eosinophils, basophils) and PBMCs/agranulocytes (monocytes, dentritic cells, T cells, B cells, NK cells)
• Platelets (thrombocytes) = the blood clotting cells, the smallest blood cell type
• Related: buffy cone refers to the leukocyte-rich waste product from plateletpheresis procedures. It isn't the same as buffy coat , which is a layer from the density gradient, but both are sources of leukocytes.

3. Red blood cells (erythrocytes): red layer at the bottom, 44-45% of blood volume. 

• Adult red blood cells are anucleated (don't have a nucleus). The nucleus is lost during cell maturation.
• Fetal red blood cells do have a nucleus . This affects cell density makes it more difficult to use density gradient medium protocols to separate cord blood into the three layers, compared to adult peripheral blood.

Learn more about blood components and blood cell types:

• Blood (human), MACS Handbook by Miltenyi Biotec. Includes useful figures and tables.
• "What is buffy coat in blood? Buffy coat preparation and buffy coat cell extraction" by Akadeum Life Science
  

Buffy coat components (leukocytes and platelets)

Cell biomarkers in placenta research

Context is important for cell markers. Before you use these markers, consider that KRT7 is a marker for trophoblast cells in placenta, but you would not say that any particular cell is a trophoblast just because it expresses KRT7. It is also a common marker for lung research even though lungs don't have any trophoblasts. That is because KRT7 is generally expressed by epithelial cells, and in placenta those are trophoblasts, but lungs have other epithelial cells. Sample context is important.

Also consider RNA vs protein, localization, and experiment use. Some markers are useful to identify cell types in single cell RNA-seq results because the RNA is specifically or mostly expressed by specific cell types, but the protein is not expressed at the cell surface so you wouldn't use that marker to sort cells for flow cytometry. 

• KRT7 = CK7 = Cytokeratin 7 = epithelial cells, trophoblast cells
• Expressed by all epithelial cells
• All trophoblast cells in placenta are epithelial, so KRT7 is used as a trophoblast cell marker in placenta tissue
• qRT-PCR marker
• Cell surface marker suitable for flow cytometry
• Beware that fibroblasts also express KRT7 and primary cells in culture will eventually become fibroblast cells

• KRT8  = epithelial cell marker, trophoblast marker
• All trophoblast cells in placenta are epithelial, so KRT8 is used as a trophoblast cell marker in placenta tissue

• CDX2 = marker for early cytotrophoblasts (CTBs)
• Expressed in CTBs around 6 weeks, but diminishes as 1st trimester progresses (Horii et al 2016).
• Indicates stem cell-like phenotypes
• Immunohistochemistry (IHC) and gene expression marker for early cytotrophoblasts

HTR8/SVneo total RNA-seq data (Lee et al 2016)

We performed a knockdown experiment with human placenta cell line HTR8/SVneo and sent N=3 samples per group for total RNA-sequencing. This RNA-seq is a good resource for people interested not only in GATA3 (our gene of interest in this experiment), but also other genes. 

Use the control samples as a resource to see roughly how well other genes are expressed in HTR8/SVneo. Beware that there may be slight expression differences between different subcultures of HTR8/SVneo due to genetic drift and culture conditions.

Citation:

Bora Lee, Lindsay L Kroener, Ning Xu, Erica T Wang, Alexandra Banks, John Williams 3rd, Mark O Goodarzi, Yii-der I Chen, Jie Tang, Yizhou Wang, Vineela Gangalapudi, Margareta D Pisarska. "Function and Hormonal Regulation of GATA3 in Human First Trimester Placentation" Biol Reprod. 2016 Nov;95(5):113. doi: 10.1095/biolreprod.116.141861. Epub 2016 Oct 12.

Placenta research 101

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